Adhesion of acidic lipid vesicles by 21.5 kDa (recombinant) and 18.5 kDa isoforms of myelin basic protein.

Myelin basic protein (MBP) is thought to be responsible for adhesion of the intracellular surfaces of compact myelin to give the major dense line. The 17 and 21.5 kDa isoforms containing exon II have been reported by others to localize to the cytoplasm and nucleus of murine oligodendrocytes and HeLa cells while the 14 and 18.5 kDa isoforms lacking exon II are confined to the plasma membrane. However, we show that the exon II(-) 18.5 kDa form and a recombinant exon II(+) 21.5 kDa isoform both caused similar aggregation of acidic lipid vesicles, indicating that they should have similar abilities to bind to the intracellular lipid surface of the plasma membrane and to cause adhesion of those surfaces to each other. The circular dichroism spectra of the two isoforms indicated that both had a similar secondary structure. Thus, both isoforms should be able to bind to and cause adhesion of the cytosolic surfaces of compact myelin. The fact that they do not could be due to differences in post-translational modification in vivo, trafficking through the cell and/or subcellular location of synthesis, but it is not due to differences in their lipid binding.

Highly deiminated isoform of myelin basic protein from multiple sclerosis brain causes fragmentation of lipid vesicles.

Myelin basic protein (MBP) occurs as a number of charge isomers due to phosphorylation, deamidation, and deimination of arginine to citrulline. All of these modifications decrease the net positive charge of the protein and its ability to cause aggregation of negatively charged lipid vesicles. This is used as a model system for the ability of MBP to cause adhesion of the cytosolic surfaces of myelin. Therefore, the effect of two deiminated forms of MBP on lipid vesicles was compared with that of the unmodified, most positively charged isomer, C1, to determine how loss of positively charged arginines would affect the function of MBP. The deiminated forms were the isomer isolated from normal human brains, in which only 6 Arg are deiminated to citrulline (MBP-Cit(6)), and an isomer isolated from the brain of a patient who died with acute, fulminating multiple sclerosis (Marburg type), in which 18 of the 19 Arg were deiminated (MBP-Cit(18)). Whereas C1 caused aggregation of lipid vesicles, resulting in an increase in absorbance due to light scattering, MBP-Cit(18) caused a decrease in absorbance of the lipid vesicles. Size exclusion chromatography and negative staining electron microscopy showed that this was due to fragmentation of the large multilayered vesicles into much smaller vesicles. MBP-Cit(6) caused less aggregation of lipid vesicles than did C1. However, no fragmentation of the vesicles into smaller ones in the presence of C1 and MBP-Cit(6) was detected by size exclusion chromatography or electron microscopy. The membrane fragmentation caused by MBP-Cit(18) is dramatically different from the effects of other forms of MBP from normal brain and may indicate a pathogenic effect of this charge isomer, which may have contributed to the severity of the Marburg type of multiple sclerosis. Alternatively, the deimination may have been a secondary effect resulting from the disease process. Regardless of the role of MBP-Cit(18) in multiple sclerosis, the effect of this modification indicates that, when most of the arginines of MBP are modified to an uncharged amino acid, the protein acquires properties similar to an apolipoprotein; thus, it may take up an amphipathic structure when bound to lipid. A partly amphipathic character may also be related to the role of MBP-Cit(6) in normal immature myelin, where it is the predominant charge isomer.

Divalent cation-mediated interaction between cerebroside sulfate and cerebrosides: an investigation of the effect of structural variations of lipids by electrospray ionization mass spectrometry.

Divalent cations mediate a carbohydrate-carbohydrate association between the two major glycolipids, galactosylceramide (GalCer) and its sulfated form, cerebroside sulfate (CBS), of the myelin sheath. We have suggested that interaction between these glycolipids on apposed extracellular surfaces of myelin may be involved in the stability or function of this multilayered structure. A mutant mouse lacking galactolipids because of a disruption in the gene that encodes a galactosyltransferase forms myelin that initially appears relatively normal but is unstable. This myelin contains glucosylceramide (GlcCer) instead of GalCer. To better understand the role of GlcCer in myelin in this mutant, we have compared the ability of divalent cations to complex CBS (galactosyl form) with GlcCer or GalCer in methanol solution by using positive ion electrospray ionization mass spectrometry. Because both the alpha-hydroxylated fatty acid species (HFA) and the nonhydroxylated fatty acid species (NFA) of these lipids occur in myelin, we have also compared the HFA and NFA species. In addition to monomeric Ca2+ complexes of all three lipids and oligomeric Ca2+ complexes of both GalCer and GlcCer, Ca2+ also caused heterotypic complexation of CBS to both GalCer and GlcCer. The heterotypic complexes had the greatest stability of all oligomers formed and survived better at high declustering potentials. Complexes of CBS with GlcCer were less stable than those with GalCer. This was confirmed by using the free sugars and glycosides making up the carbohydrate headgroups of these lipids. HFA species of CBS and GalCer formed more stable complexes than NFA species, but hydroxylation of the fatty acid of GlcCer had no effect. The ability of GlcCer to also complex with CBS, albeit with lower stability, may allow GlcCer to partially compensate for the absence of GalCer in the mouse mutant.

Analysis of the membrane-interacting domains of myelin basic protein by hydrophobic photolabeling.

Myelin basic protein is a water soluble membrane protein which interacts with acidic lipids through some type of hydrophobic interaction in addition to electrostatic interactions. Here we show that it can be labeled from within the lipid bilayer when bound to acidic lipids with the hydrophobic photolabel 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine (TID) and by two lipid photolabels. The latter included one with the reactive group near the apolar/polar interface and one with the reactive group linked to an acyl chain to position it deeper in the bilayer. The regions of the protein which interact hydrophobically with lipid to the greatest extent were determined by cleaving the TID-labeled myelin basic protein (MBP) with cathepsin D into peptides 1-43, 44-89, and 90-170. All three peptides from lipid-bound protein were labeled much more than peptides from the protein labeled in solution. However, the peptide labeling pattern was similar for both environments. The two peptides in the N-terminal half were labeled similarly and about twice as much as the C-terminal peptide indicating that the N-terminal half interacts hydrophobically with lipid more than the C-terminal half. MBP can be modified post-translationally in vivo, including by deamidation, which may alter its interactions with lipid. However, deamidation had no effect on the TID labeling of MBP or on the labeling pattern of the cathepsin D peptides. The site of deamidation has been reported to be in the C-terminal half, and its lack of effect on hydrophobic interactions of MBP with lipid are consistent with the conclusion that the N-terminal half interacts hydrophobically more than the C-terminal half. Since other studies of the interaction of isolated N-terminal and C-terminal peptides with lipid also indicate that the N-terminal half interacts hydrophobically with lipid more than the C-terminal half, these results from photolabeling of the intact protein suggest that the N-terminal half of the intact protein interacts with lipid in a similar way as the isolated peptide. The similar behavior of the intact protein to that of its isolated peptides suggests that when the purified protein binds to acidic lipids, it is in a conformation which allows both halves of the protein to interact independently with the lipid bilayer. That is, it does not form a hydrophobic domain made up from different parts of the protein.

Proton NMR study of peptides from myelin basic protein: evidence for Lys74-His77 interaction revealed from histidine line broadening.

Residues 69-84 of guinea pig myelin basic protein contain the encephalitogenic determinant for the Lewis rat. Insertion of histidine and glycine at positions 77 and 78 in bovine MBP greatly reduces the encephalitogenicity of the protein. Synthetic peptides analogous to this region of MBP containing glycine and histidine are encephalitogenic if they lack the N-terminal half, residues 69-74. However, if they contain both histidine plus the N-terminal half, encephalitogenicity is abolished, suggesting that an interaction of histidine with an amino acid in the N-terminal half changes the conformation or the properties of the peptide. This was investigated by measuring the 1H-NMR spectra of synthetic peptides analogous to this region of MBP, both containing histidine but with and without the N-terminal half. The major difference in the spectra of the two peptides was the pH dependence of line broadening of the histidine resonances. The histidine C2H and C4H resonances were broadened at intermediate pH values in both peptides. However, sharpening of the lines at high pH showed a different pH dependence in the two peptides. For the longer peptide containing the N-terminal half, the lines did not sharpen until the pH was increased above 10.2, coinciding with the pKa of Lys-74. Acetylation of this peptide caused the pH at which the lines began to sharpen to drop to 8.8. In the shorter peptide, lacking the N-terminal half and Lys-74, the lines also sharpened at pH 8.8. The greater broadening which persisted up above pH 10 for the longer peptide suggests slow exchange between two different conformations or environments of the histidine. One of these could be a conformation in which the deprotonated histidine hydrogen bonds with Lys-74. The Lys side-chain resonances indicated a decrease in rotational freedom above the pKa of histidine, consistent with this conclusion. Although this putative interaction between His and Lys-74 did not appear to have a significant effect on the overall conformation of the peptide, it could result in a reduction in encephalitogenicity by altering the properties of the peptide. This could affect processing and presentation of this determinant by antigen presenting cells.

Investigation of the calcium-mediated association between the carbohydrate head groups of galactosylceramide and galactosylceramide I3 sulfate by electrospray ionization mass spectrometry.

Calcium has been shown previously to cause aggregation of phosphatidylcholine/cholesterol liposomes containing galactosylceramide (GalCer) with similar liposomes containing cerebroside sulfate (galactosylceramide I3 sulfate) (CBS), suggesting that it mediates a carbohydrate-carbohydrate association between these two glycolipids. In order to determine if such an association occurs, the noncovalent complexes formed on addition of calcium chloride to GalCer and CBS in methanol were examined by positive and negative ion spray mass spectrometry. Monomeric Ca2+ complexes of both lipids were observed. In addition, Ca2+ also caused oligomerization of GalCer. Oligomerization of CBS anion was not seen, but dimers would not have been observed, as they would be neutral. However, Ca2+ caused heterotypic complexation of GalCer and CBS. Although these heterotypic complexes were of low abundance in methanol compared with the other monomeric and homotypic oligomeric positive ions formed at low declustering potentials, the heterotypic dimer [GalCer.CBS.Ca2+-H]+ had the greatest stability of all oligomers formed and was the only one to survive at high declustering potentials. Na+ did not cause oligomerization of GalCer in methanol indicating that the complexes of GalCer with Ca2+ are not caused by van der Waals interactions between the lipid moieties. GalCer and CBS are present in high concentrations in myelin. This Ca2+-mediated carbohydrate-carbohydrate interaction, which can bridge apposing bilayers, may be involved in adhesion of the extracellular surfaces of the myelin sheath.

Do the long fatty acid chains of sphingolipids interdigitate across the center of a bilayer of shorter chain symmetric phospholipids?

Novel cerebroside sulfate (CBS) spin labels containing long chain C24 or C26 fatty acids with a nitroxide spin label on the 22nd carbon were synthesized and used to investigate the ability of the long fatty acid chains of glycosphingolipids to interdigitate across the center of a non-interdigitated bilayer of phospholipids formed of symmetric saturated or unsaturated shorter fatty acid chain species, in the presence or absence of cholesterol. The motion of these long chain spin labels incorporated at 1 mole% in dimyristoylphosphatidylcholine (diC14-PC), dipalmitoylphosphatidylcholine (diC16-PC), distearoylphosphatidylcholine (diC18-PC), dibehenoylphosphatidylcholine (diC22-PC), spingomyelin (SM), 1-stearoyl-2-oleoylphosphatidylcholine (18:0.18:1-PC), and dimyristoylphosphatidylethanolamine (diC14-PE) was compared to that of CBS spin labels containing stearic acid spin labeled at the 5th carbon and at the 16th carbon. The results indicated that the C26 chain is interdigitated in the gel phase of diC14-PC, diC16-PC, SM, and possibly diC18-PC, but not diC14-PE, and the C24 chain may interdigitate in diC14-PC but not in the other phospholipids. Thus in order to interdigitate across the center of gel phase bilayers, the long acyl chain of the sphingolipid probably must be long enough to nearly span the phospholipid bilayer. The inability to interdigitate in diC14-PE is likely due to the close packing of this lipid in the gel phase. The C26 chain may also be interdigitated in these lipids in the presence of cholesterol at low temperatures. However, at physiological temperatures in the presence of cholesterol and in the liquid-crystalline phase of all the lipids, the results indicate that the long acyl chain of the glycosphingolipid is not interdigitated, but rather must terminate at the bilayer center. This may force the carbohydrate headgroup of the glycosphingolipid farther above the bilayer surface, allowing it to be recognized better by various carbohydrate binding ligands and proteins.

Thermotropic phase behavior of mixtures of long chain fatty acid species of cerebroside sulfate with different fatty acid chain length species of phospholipid.

The thermotropic phase behavior of asymmetric, long fatty acid chain species of cerebroside sulfate, C24-CBS and C26-CBS, with symmetric species of phosphatidylcholine (PC) containing fatty acid chains of 14-18 carbons in length (diC14-PC, diC16-PC, diC18-PC) and dimyristoylphosphatidylethanolamine (diC14-PE) in 0.1 M KCl was studied by differential scanning calorimetry. Novel cerebroside sulfate (CBS) spin labels containing long chain C24 and C26 fatty acid spin labels with the nitroxide group on the twenty-second carbon were used to study the lipid organization of the gel phases of these mixtures. The phase diagrams of all the mixtures indicated the presence of two immiscible gel phases at low CBS concentrations. All except the C26-CBS/diC14-PC mixture had eutectic phase behavior at low CBS concentrations suggesting that the long fatty acid chain of the CBS species had a destabilizing effect on the gel phase of most of the phospholipids. The C26-CBS/diC14-PC mixture had peritectic phase behavior at low CBS concentrations indicating a stabilizing effect of the CBS C26 acyl chain on diC14-PC. These results are consistent with the relative compatibility of the CBS acyl chain length with the bilayer thickness of the PC; only in the case of the C26-CBS/diC14-PC mixture is the acyl chain of CBS long enough to span the PC bilayer. At intermediate to high CBS concentrations, the CBS and phospholipid (PL) were miscible with the exception of the C24-CBS/diC18-PC combination, which had eutectic phase behavior over a wide concentration range. Thus when the PL acyl chain length was similar to the sphingosine chain length of CBS, CBS bilayers could accommodate symmetric phospholipid molecules better than phospholipid bilayers could accommodate asymmetric molecules of CBS. Use of the spin labels indicated that, at low temperatures and at intermediate to high CBS concentrations, all of the mixtures were in a triple chain mixed interdigitated gel phase which immobilized the spin label. This gel phase slowly transformed over a wide temperature range to a double chain partially interdigitated gel phase in which the spin labels had much more motion. This transformation could be detected as a broad low enthalpy transition by differential scanning calorimetry. In all cases the presence of phospholipid destabilized the mixed interdigitated phase. Stabilization of the partially interdigitated bilayer by intermolecular hydrogen bonding interactions must outweigh the destabilizing forces caused by disruptions in packing and van der Waals interactions between CBS molecules resulting from insertion of molecules of phospholipid into this type of bilayer.(ABSTRACT TRUNCATED AT 400 WORDS)

Interference of carbohydrates in the quantitation of protein-bound citrulline by amino acid analysis.

After cation-exchange chromatographic separation of the charge isomers of human myelin basic protein, the citrulline-containing component was purified from the unbound fraction by gel permeation chromatography. Sephadex G75 (superfine) resolved high- and low-molecular-weight contaminants from the 18.5K myelin basic protein. However, carbohydrates leached from the column material by the acidic eluant interfered with citrulline quantitation by amino acid analysis. It appears highly probable that during acid hydrolysis of the protein, glucose reacts with citrulline, the ureido group of the latter forming a condensation product with the sugar which subsequently undergoes further chemical degradation to products which are not easily identifiable. Methods for removing the interfering sugars prior to amino acid analysis are discussed.

Mixtures of semisynthetic species of cerebroside sulfate with dipalmitoyl phosphatidylcholine. Thermotropic phase behavior and permeability.

The phase behavior of mixtures of dipalmitoyl phosphatidylcholine (DPPC) with semisynthetic species of cerebroside sulfate (CBS) containing palmitic acid (C16:0-CBS) or lignoceric acid (C24:0-CBS) in 0.1 M KCl was studied using differential scanning calorimetry. DPPC and C16:0-CBS were miscible in all proportions in the gel phase above 10 mol% CBS and in the liquid-crystalline phase. However, C24:0-CBS was less miscible with DPPC over a wide concentration range in the gel phase. At high CBS concentrations it was probably also not entirely miscible with DPPC in the liquid-crystalline phase. Small amounts of both species of CBS lowered the transition temperature and enthalpy of DPPC, suggesting that they are more soluble in the liquid-crystalline phase of DPPC than the gel phase. The transition temperature at higher CBS concentrations was also less than expected, especially after cycling through the phase transition in the case of C24:0-CBS, suggesting that mixing with DPPC inhibited the intermolecular hydrogen bonding interactions and dehydration of CBS. In C24:0-CBS-DPPC mixtures several populations were present over a wide compositional range, including two solid-solid solutions of fixed composition. At high C24:0-CBS concentrations some C24:0-CBS also phase separated out of the mixture. Structural considerations suggested that the C24:0-CBS which is mixed with DPPC must be interdigitated into the DPPC bilayer. Other populations that are present may have a different structural organization. A fatty acid spin label in these mixtures was a little less ordered than in either lipid by itself. The permeability of these lipids, as well as the two asymmetric species 1-stearoyl-2-caproyl phosphatidylcholine and 1-stearoyl-2-myristoyl phosphatidylcholine (18:10PC and 18:14PC), to a water-soluble spin label tempocholine chloride was also measured. The studies with 18:10PC and 18:14PC indicated that both triple-chain mixed interdigitated bilayers and double-chain partially interdigitated bilayers can trap water-soluble substances and have low permeability. Both species of CBS could also entrap the spin label and had low permeability at 4 degrees C. However, they rapidly lost the entrapped compound when they transformed into their stable dehydrated phases or into the liquid-crystalline phase. Mixing with DPPC prevented both of these losses. These studies supported the conclusion that a significant amount of the CBS was mixed with the DPPC and that this mixing prevented the dehydration changes which CBS undergoes by itself.(ABSTRACT TRUNCATED AT 400 WORDS)

Influence of structural modifications on the phase behavior of semi-synthetic cerebroside sulfate.

Cerebroside sulfate (galactosylceramide I3-sulfate) containing alpha-hydroxy lignoceric acid (C24:0h-CBS), nervonic acid (C24:1-CBS), or hexacosanoic acid (C26:0-CBS) was prepared by a semi-synthetic procedure and studied by differential scanning calorimetry. The phase behavior of these species in 2 M KCl was compared to that of shorter chain length hydroxy and non-hydroxy fatty acid species reported earlier. All three of the new lipids undergo metastable phase behavior similar but not identical to the other species. In addition, the metastable phase behavior of all of the non-hydroxy fatty acid species was found to be more complex than previously thought, with several phases of high transition temperatures and enthalpies possible. Fatty acid hydroxylation inhibits the transition from the metastable to some of the more stable phases. It also significantly increases the phase transition temperatures of both the metastable and stable phases indicating that it contributes to the hydrogen bonding network formed between the lipid molecules and helps overcome the lateral repulsive effect of the negatively charged sulfate. The C-15 cis double bond significantly lowers the temperature and enthalpy of the phase transition indicating that it increases the lateral separation of the lipid molecules and decreases the intermolecular hydrogen bonding interactions. However, it does not prevent formation of a more stable phase. By comparing the effect of various structural modifications reported here and earlier it could be concluded that fatty acid chain length has little effect on the phase transition temperature and enthalpy. This suggests that the forces between the lipid molecules may be dominated by head group interactions rather than interactions between the lipid chains. However, fatty acid chain length has a significant effect on the tendency of the hydroxy fatty acid species to form the more stable phase. The ease of formation of the stable phase increases with increase in chain length. Thus an increase in chain length helps overcome the kinetic barrier to stable phase formation presented by hydroxylation of the fatty acid.

Interdigitated lipid bilayers of long acyl chain species of cerebroside sulfate. A fatty acid spin label study.

The metastable phase behavior of semi-synthetic species of cerebroside sulfate (CBS), with hydroxy and non-hydroxy fatty acids from 16 to 26 carbons in length, was compared in Li+ and K+ using differential scanning calorimetry. The structure of the metastable and various stable phases formed in the presence of these two cations was investigated using a fatty acid spin label, 16-doxylstearate. A number of stable phases with successively higher phase transition temperatures and enthalpies occur in the presence of K+ (see the preceding paper). Li+ prevents formation of the most stable phases with the highest transition temperatures and enthalpies for all species of CBS. However, it does not prevent a transition from the metastable phase to the first stable phase of the longer chain C24 and C26 species. Furthermore, it allows C24:0h-CBS to undergo a similar transition, in contrast to a high K+ concentration, which prevents it. The spin label has anisotropic motion in the metastable gel phase formed by all species of CBS on cooling from the liquid crystalline phase. The spectra resemble those in gel phase phospholipids. The spin label is partially insoluble in the most stable phases formed by all the lipids, including the unsaturated C24:1 species, preventing further elucidation of their structure using this technique. However, the spin label is soluble in the first stable phase formed on cooling by the longer chain C24:0 and C26:0-CBS in Li+ and K+ and by C24:0h-CBS in Li+, and is motionally restricted in this phase. The motional restriction is similar to that observed in the mixed interdigitated bilayers of asymmetric species of phosphatidylcholine and fully interdigitated bilayers formed by symmetric phospholipids. It strongly suggests that the highly asymmetric long chain species of CBS form a mixed interdigitated bilayer in their first stable gel phases while the metastable phase of these and the shorter chain lipids may be partially interdigitated. The metastable phase of C24:1-CBS is more disordered suggesting that it may not be interdigitated at all. Thus the results suggest that (i) the hydroxy fatty acid inhibits but does not prevent formation of a mixed interdigitated bilayer by long chain species of CBS, (ii) an increase in non-hydroxy fatty acid chain length from 24 to 26 carbons promotes it, and (iii) a cis double bond probably prevents any form of interdigitation. These results may be relevant to the physiological and pathological roles of these structural modifications of CBS.

Photolabeling of myelin basic protein in lipid vesicles with the hydrophobic reagent 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine.

The hydrophobic photolabel 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine([125I]TID) was used to label myelin basic protein or polylysine in aqueous solution and bound to lipid vesicles of different composition. Although myelin basic protein is a water soluble protein which binds electrostatically only to acidic lipids, unlike polylysine it has several short hydrophobic regions. Myelin basic protein was labeled to a significant extent by TID when in aqueous solution indicating that it has a hydrophobic site which can bind the reagent. However, myelin basic protein was labeled 2-4-times more when bound to the acidic lipids phosphatidylglycerol, phosphatidylserine, phosphatidic acid, and cerebroside sulfate than when bound to phosphatidylethanolamine, or when in solution in the presence of phosphatidylcholine vesicles. It was labeled 5-7-times more than polylysine bound to acidic lipids. These results suggest that when myelin basic protein is bound to acidic lipids, it is labeled from the lipid bilayer rather than from the aqueous phase. However, this conclusion is not unequivocal because of the possibility of changes in the protein conformation or degree of aggregation upon binding to lipid. Within this limitation the results are consistent with, but do not prove, the concept that some of its hydrophobic residues penetrate partway into the lipid bilayer. However, it is likely that most of the protein is on the surface of the bilayer with its basic residues bound electrostatically to the lipid head groups.

Antigen-targeted liposome-encapsulated methotrexate specifically kills lymphocytes sensitized to the nonapeptide of myelin basic protein.

Antigen targeting of liposome-encapsulated cytotoxic drugs to specific lymphocytes may be a useful approach for antigen-specific immunosuppressive treatment of autoimmune diseases in which a specific antigen is involved. The feasibility of utilizing this approach was investigated using experimental allergic encephalomyelitis as an animal model for an autoimmune response. The encephalitogenic determinant of myelin basic protein for the guinea pig is contained in residues 114-122, the so-called nonapeptide. We have acylated the nonapeptide at its N-terminal to anchor it in the lipid bilayer of liposomes containing the cytotoxic drug methotrexate. The nonapeptide on the surface of the liposomes then allows targeting of the liposomal methotrexate in vitro to anti-nonapeptide T lymphocytes obtained from guinea pigs with experimental allergic encephalomyelitis. Treatment with the nonapeptide-targeted liposomal methotrexate inhibited proliferation of anti-nonapeptide lymphocytes significantly more than that of control lymphocytes. These included non-sensitized lymphocytes, stimulated with phytohemagglutinin, or lymphocytes sensitized to different, unrelated proteins, the purified protein derivative of tuberculin and keyhole limpet hemocyanin, and stimulated with their specific antigens. Furthermore, nonapeptide-targeted liposomes had a greater cytotoxic effect on anti-nonapeptide T cells than untargeted liposomes. The results indicated that specific targeting to and killing of anti-nonapeptide cells was achieved, although improvements of the treatment are necessary before its use can be attempted in vivo.

Factors affecting surface expression of glycolipids: influence of lipid environment and ceramide composition on antibody recognition of cerebroside sulfate in liposomes.

The reactivity of the acidic glycolipid cerebroside sulfate (CBS) with antibody was studied as a function of its lipid environment in vesicles and of its ceramide composition. The lipid environment was varied by using phosphatidylcholine of varying chain length with cholesterol in a phosphatidylcholine:cholesterol:cerebroside sulfate molar ratio to glycolipid of 1:0.75:0.1. The ceramide structure of CBS was varied by using synthetic forms containing palmitic acid, lignoceric acid, or the corresponding alpha-hydroxy fatty acids. Reactivity with antibody was determined by measuring complement-mediated lysis of the vesicles containing a spin-label marker, tempocholine chloride. The data were analyzed by a theoretical model which gives relative values for the dissociation constant and concentration of antibodies within the antiserum which are able to bind to the glycolipid. If the phosphatidylcholine chain length was increased, increasing the bilayer thickness, only a small population of high-affinity antibodies were able to bind to cerebroside sulfate, suggesting decreased surface exposure of the glycosyl head group. A larger population of lower affinity antibodies were able to bind to it in a shorter chain length phosphatidylcholine environment. However, if the chain length of the cerebroside sulfate was increased, it could be recognized by more antibodies of lower affinity than the short chain length form, suggesting that an increase in chain length of the glycolipid increased surface exposure. Hydroxylation of the fatty acid inhibited antibody binding; only a smaller population of higher affinity antibodies was able to bind to the hydroxy fatty acid forms.(ABSTRACT TRUNCATED AT 250 WORDS)

Interaction of myelin basic protein and polylysine with synthetic species of cerebroside sulfate.

The effect of myelin basic protein on the myelin lipid cerebroside sulfate was studied by differential scanning calorimetry and use of the fatty acid spin label, 16-S-SL, in order to determine (i) the effect of basic protein on the metastable phase behavior experienced by this lipid, and (ii) to determine if basic protein perturbs the lipid packing as it does with some acidic phospholipids. The effects of basic protein on the thermodynamic parameters of the lipid phase transition were compared with those of polylysine which has an ordering effect on acidic phospholipids as a result of its electrostatic interactions with the lipid head groups. Different synthetic species of cerebroside sulfate of varying fatty acid chain length and with and without a hydroxy fatty acid were used. The non-hydroxy fatty acid forms of cerebroside sulfate undergo a transition from a metastable to a more ordered stable state while the hydroxy fatty acid forms remain in the metastable state at the cation concentration used in this study (0.01 M Na+ or K+). The non-hydroxy fatty acid forms were still able to go into a stable state in the presence of both basic protein and polylysine. At low concentrations, basic protein increased the rate of the transition to the stable state, while polylysine decreased it for the longest chain length form studied. However, at high concentrations, basic protein probably prevented formation of the stable state. The hydroxy fatty acid forms did not go into the stable state in the presence of basic protein and polylysine. It is argued that the increased rate of formation of the stable state in the presence of basic protein and decreased rate in the presence of polylysine are consistent with interdigitation of the lipid acyl chains in the stable state. Basic protein also had a small perturbing effect on the lipid. It decreased the total enthalpy of the lipid phase transition. When added to the non-hydroxy fatty acid forms it increased the temperature of the liquid crystalline to metastable phase transition and decreased the temperature of the stable to liquid crystalline phase transition. It significantly decreased the transition temperature of the hydroxy fatty acid forms but only a portion of the lipid was affected. In contrast, polylysine increased the transition temperature of the metastable and stable states of all forms of cerebroside sulfate but had a greater effect on the non-hydroxy fatty acids forms than on the hydroxy fatty acid forms.(ABSTRACT TRUNCATED AT 400 WORDS)

Partial synthesis and physical properties of cerebroside sulfate containing palmitic acid or alpha-hydroxy palmitic acid.

Chromatographically pure galactosylceramide I3-sulfate (cerebroside sulfate (CBS)) containing palmitic acid or D-2-hydroxypalmitic acid has been prepared by the acylation of galactosylsphingosine I3-sulfate obtained from the saponification of bovine brain sulfatides. Optically pure D-2-hydroxypalmitic acid was obtained by adapting literature methods for the synthesis of the racemic acid and its resolution. The thermotropic behavior of the two synthetic CBSs were compared to each other and to the corresponding components in natural CBS, obtained by fractionation of bovine brain sulfatides, in order to determine the contribution of the hydroxy fatty acid to intermolecular hydrogen bonding between molecules of the lipid. The gel to liquid crystalline phase transition temperature (Tc) of the hydroxy fatty acid (HFA) synthetic form is 53.2 degrees C, 3 degrees higher than that of the non-hydroxy fatty acid (NFA) form at low concentrations of Na+ or K+. A similar difference was found for the HFA and NFA forms of natural CBS. The enthalpy of the NFA synthetic form is 8.5 kcal/mol, about 30% greater than that of the HFA form. The difference in Tc between the NFA and HFA forms is abolished as the Na+ or K+ concentration increases but the difference in enthalpy persists. Increasing cation concentration, over the range 0.01-2 M, increases Tc more than for an acidic phospholipid, phosphatidylglycerol, probably due to increased intermolecular hydrogen bonding as the charged sulfate is shielded. K+ causes a 3-4 degrees C greater increase in Tc relative to that produced by Na+ while K+ and Na+ have similar effects on phosphatidylglycerol.

A high-yield saponification of galactosylceramide I(3)-sulfate.

A method for the deacylation of galactosylceramide I(3)-sulphate using aqueous methanolic KOH is described. The combination of a relatively low concentration of the alkali (0.3 M) and a moderate reaction temperature (reflux point of 90% methanol) results in the formation of galactosylsphingosine I(3)-sulphate in consistently high yields (61%) with a minimum of side reactions. The product was purified by column chromatography and its identity established by thin layer chromatography, infrared spectroscopy, determination of galactose content and organic sulphate assay using established methods or their modifications.

Thallium ingestion with survival: ineffectiveness of peritoneal dialysis and potassium chloride diuresis.

A 52-year-old man who ingested 20 g of thallium iodide and survived is reported. Therapy included gastrointestinal decontamination, peritoneal dialysis, and potassium chloride diuresis. Peritoneal dialysis removed 4% of the calculated ingested dose in a 24-h period and potassium chloride diuresis 2%, an insignificant amount in light of the ingested dose of thallium. The primary method of treatment of thallium poisoning remains prompt gastrointestinal decontamination.