Predictors of catheter-related gram-negative bacilli bacteraemia among cancer patients.

Gram-negative bacillary bacteraemia (GNB) is associated with high morbidity and mortality among cancer patients. We conducted this study to determine the risk factors that may predict the catheter as the source of GNB in cancer patients. From July 2005 to December 2006 all 266 cancer patients with GNB and central venous catheters (CVCs) at The University of Texas M. D. Anderson Cancer Centre in Houston, were classified as catheter-related bloodstream infection (CRBSI) according to Infectious Diseases Society of America criteria. We compared clinical and microbiological features of CRBSIs and non-CRBSIs. We identified 78 CRBSIs and 126 non-CRBSIs. On univariate analysis, polymicrobial bacteraemia, Stenotrophomonas maltophilia bacteraemia, and more than 1000 CFUs in CVC blood cultures, were more common among CRBSI cases. Escherichia coli bacteraemia, haematologic cancer, neutropenia and prior antibiotic use were more common among non-CRBSI cases. On multivariate analysis, S. maltophilia bacteraemia (odds ratio (OR), 5.78; 95% confidence interval (CI), 1.47-22.78; p 0.045), polymicrobial bacteraemia (OR, 4.04; 95% CI, 1.56-10.44; p 0.042), and more than 1000 CFUs from CVC blood cultures (OR, 4.39; 95% CI, 2.02-9.27; p <0.01), were associated with CRBSI. Neutropenia was associated with non-CRBSI (OR, 0.26; 95% CI, 0.13-0.53; p <0.01). Several factors such as S. maltophilia bacteraemia, polymicrobial bacteraemia and more than 1000 CFUs from a blood culture drawn through the CVC may assist the clinicians in assessing whether an indwelling catheter is the source of a GNB and hence CVC removal may be considered.

The evolving role of high-density lipoprotein in reducing cardiovascular risk.

In many patients with coronary artery disease, a low level of high-density lipoprotein cholesterol (HDL-C), rather than substantially elevated low-density lipoprotein cholesterol (LDL-C), is often the predominant lipid abnormality. Although the National Cholesterol Education Program treatment guidelines include HDL-C concentration as a major risk factor for primary prevention, the guidelines' emphasis on LDL-C as the primary target of therapy may cause uncertainty as to whether risk reduction strategies should focus on lowering LDL-C or raising HDL-C in high-risk patients with low HDL-C. Recent clinical trial evidence and epidemiologic data suggest that HDL-C should play a more important role in risk assessment, and that the definition of low HDL-C may need adjustment from the current National Cholesterol Education Program definition of <35 mg/dL to perhaps <40 mg/dL in men and <45 mg/dL in women. Patients with low HDL-C should receive aggressive risk factor modification, and more emphasis on increasing HDL-C may be warranted in addition to lowering LDL-C. (c) 2001 by CHF, Inc.

Interaction of lipid-bound myelin basic protein with actin filaments and calmodulin.

Myelin basic protein (MBP) binds to negatively charged lipids on the cytosolic surface of oligodendrocytes (OLs) and is believed to be responsible for adhesion of these surfaces in the multilayered myelin sheath. MBP in solution has been shown by others to bind to both G- and F-actin, to bundle F-actin filaments, and to induce polymerization of G-actin. Here we show that MBP bound to acidic lipids can also bind to both G- and F-actin and cause their sedimentation together with MBP-lipid vesicles. Thus it can simultaneously utilize some of its basic residues to bind to the lipid bilayer and some to bind to actin. The amount of actin bound to the MBP-lipid vesicles decreased with increasing net negative surface charge of the lipid vesicles. It was also less for vesicles containing the lipid composition predicted for the cytosolic surface of myelin than for PC vesicles containing a similar amount of an acidic lipid. Calmodulin caused dissociation of actin from MBP and of the MBP-actin complex from the vesicles. However, it did not cause dissociation of bundles of actin filaments once these had formed as long as some MBP was still present. These results suggest that MBP could be a membrane actin-binding protein in OLs/myelin and its actin binding can be regulated by calmodulin and by the lipid composition of the membrane. Actin binding to MBP decreased the labeling of MBP by the hydrophobic photolabel 3-(trifluoromethyl)-3-(m-[(125)I]iodophenyl)diazirine (TID), indicating that it decreased the hydrophobic interactions of MBP with the bilayer. This change in interaction of MBP with the bilayer could then create a cytosol to membrane signal caused by changes in interaction of the cytoskeleton with the membrane.

Trans interactions between galactosylceramide and cerebroside sulfate across apposed bilayers.

The two glycosphingolipids galactosylceramide (GalC) and its sulfated form, cerebroside sulfate (CBS), are present at high concentrations in the multilayered myelin sheath and are involved in carbohydrate-carbohydrate interactions between the lipid headgroups. In order to study the structure of the complex of these two glycolipids by Fourier transform infrared (FTIR) spectroscopy, GalC dispersions were combined with CBS dispersions in the presence and absence of Ca(2+). The FTIR spectra indicated that a strong interaction occurred between these glycolipids even in the absence of Ca(2+). The interaction resulted in dehydration of the sulfate, changes in the intermolecular hydrogen bonding interactions of the sugar and other oxygens, decreased intermolecular hydrogen bonding of the amide C==O of GalC and dehydration of the amide region of one or both of the lipids in the mixture, and disordering of the hydrocarbon chains of both lipids. The spectra also show that Ca(2+) interacts with the sulfate of CBS. Although they do not reveal which other groups of CBS and GalC interact with Ca(2+) or which groups participate in the interaction between the two lipids, they do show that the sulfate is not directly involved in interaction with GalC, since it can still bind to Ca(2+) in the mixture. The interaction between these two lipids could be either a lateral cis interaction in the same bilayer or a trans interaction between apposed bilayers. The type of interaction between the lipids, cis or trans, was investigated using fluorescent and spin-label probes and anti-glycolipid antibodies. The results confirmed a strong interaction between the GalC and the CBS microstructures. They suggested further that this interaction caused the CBS microstructures to be disrupted so that CBS formed a single bilayer around the GalC multilayered microstructures, thus sequestering GalC from the external aqueous phase. Thus the CBS and GalC interacted via a trans interaction across apposed bilayers, which resulted in dehydration of the headgroup and interface region of both lipid bilayers. The strong interaction between these lipids may be involved in stabilization of the myelin sheath.

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.

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.

Effect of posttranslational modifications to myelin basic protein on its ability to aggregate acidic lipid vesicles.

When isolated from central nervous system myelin, myelin basic protein (MBP) exhibits charge microheterogeneity due to posttranslational deamidation, phosphorylation, and deimination of arginine to citrulline. These modifications are known to decrease the ability of MBP to aggregate acidic lipid vesicles and thus could regulate the ability of MBP to mediate adhesion between the intracellular surfaces of myelin. The effects of salt (KCl) concentration and the protein to lipid ratio on the ability of charge isomers of MBP to aggregate large unilamellar vesicles (LUVs) were investigated. Increased salt concentration from 10 to 100 mM caused increasing aggregation of LUVs by low concentrations of all charge isomers but did not eliminate the differences in their abilities to aggregate. All isomers were bound equally up to about 100 mM K+ but were dissociated at higher K+ concentrations. The degree of dissociation increased with increasing net negative charge of the isomer. At high concentrations all charge isomers except the form in which six arginine residues are converted to citrulline (C8) aggregated LUVs of phosphatidylcholine/phosphatidylserine (PC/PS) 8:2 (mol/mol) similarly and salt increased the aggregation to the same degree for all. There was less difference in the ability of the charge isomers, including C8, to aggregate LUVs with a lipid composition resembling that of the cytoplasmic leaflet of myelin (Cyt-LUVs) than for PC/PS LUVs. Furthermore, high salt concentrations (400 mM) did not dissociate any of the charge isomers from the Cyt-LUVs. These results suggest that the reason for inhibition of aggregating ability by charge modification is not increased charge repulsion of the protein but rather its reduced multivalency of net positive charge. They indicate further that the lipid composition of the cytoplasmic leaflet is ideally suited to permit MBP-mediated adhesion and that charge modifications of MBP would probably not affect adhesion of the intracellular surfaces of compact myelin where MBP concentration is high. However, charge modifications might affect adhesion in cytoplasm-containing regions of myelin such as the paranodal loops, where MBP concentration is low and where K+ concentration may vary in the range of 60-75 mM.

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)

Interdigitation of phosphatidylcholine and phosphatidylethanolamine mixed with complexes of acidic lipids and polymyxin B or polymyxin B nonapeptide.

A fatty acid spin label, 16-doxyl-stearic acid, was used to determine the percent interdigitated lipid in mixtures of a neutral phospholipid and an acidic phospholipid. Interdigitation of the acidic lipid was induced with polymyxin B (PMB) at a mole ratio of PMB to acidic lipid of 1:5. This compound does not bind significantly to neutral lipids or induce interdigitation of the neutral lipids by themselves. The neutral lipids used were dimyristoylphosphatidylcholine (DMPC), dipalmitoylphosphatidylcholine (DPPC), or dipalmitoylphosphatidylethanolamine (DPPE), and the acidic lipids were dipalmitoylphosphatidylglycerol (DPPG) or dipalmitoylphosphatidic acid (DPPA). The percent interdigitated lipid was determined from the percent of the spin label which is motionally restricted, assuming that the spin label is homogeneously distributed in the lipid. Assuming further that 100% of the acidic lipid is interdigitated at this saturating concentration of PMB, the percentage of the neutral lipid which can become interdigitated along with it was calculated. The results indicate that about 20 mole % DPPC can be incorporated into and become interdigitated in the interdigitated bilayer of PMB/DPPG at 4 degrees C. As the temperature approaches the phase transition temperature, the lipid becomes progressively less interdigitated; this occurs to a greater degree for the mixtures than for the single acidic lipid. Thus the presence of DPPC promotes transformation of the acidic lipid to a non-interdigitated bilayer at higher temperatures. At the temperature of the lipid phase transition little or none of the lipid in the mixture is interdigitated. Thus the lipid phase transition detected by calorimetry is not that of the interdigitated bilayer. The shorter chain length DMPC can be incorporated to a greater extent than DPPC, 30-50 mol%, in the interdigitated bilayer of PMB-DPPG. This may be a result of reduced exposure of the terminal methyl groups of the shorter myristoyl chains at the polar/apolar interface of the interdigitated bilayer. Less than 29% of the total lipid was interdigitated in a DPPC/DPPA/PMB 1:1:0.2 mixture indicating that none of the DPPC in this mixture becomes interdigitated. This is attributed to the lateral interlipid hydrogen bonding interactions of DPPA which inhibits formation of an interdigitated bilayer. DPPE was found to be incorporated into the interdigitated bilayer of PMB-DPPG to a similar extent as DPPC if the amount of PMB added is sufficient to bind to only the DPPG in the mixture. Differential scanning calorimetry showed that the remaining non-interdigitated DPPE-enriched mixture phase separates into its own domain.(ABSTRACT TRUNCATED AT 400 WORDS)

Behavior of spin labels in a variety of interdigitated lipid bilayers.

The behavior of a number of spin labels in several lipid bilayers, shown by X-ray diffraction to be interdigitated, has been compared in order to evaluate the ability of the spin label technique to detect and diagnose the structure of lipid bilayers. The main difference between interdigitated and non-interdigitated gel phase bilayers which can be exploited for determination of their structure using spin labels, is that the former have a much less steep fluidity gradient. Thus long chain spin labels with the nitroxide group near the terminal methyl of the chain, such as 16-doxylstearic acid, its methyl ester, or a phosphatidylglycerol spin label containing 16-doxylstearic acid (PG-SL), are more motionally restricted and/or ordered in the interdigitated bilayer than in the non-interdigitated bilayer. This difference is large enough to be of diagnostic value for all three spin labels in the interdigitated bilayers of dihexadecylphosphatidylcholine, dipalmitoylphosphatidylcholine/ethanol, and 1,3-dipalmitoylphosphatidylcholine. However, it is not large enough to be of diagnostic value at low temperatures. Use of probes with the nitroxide group closer to the apolar/polar interface reveals that these latter interdigitated bilayers are more disordered or less closely packed. As the temperature is increased, however, the motion of the PG-SL does not increase as much in these interdigitated bilayers as in non-interdigitated bilayers. The difference in the motion and/or order of PG-SL between interdigitated and non-interdigitated bilayers is large enough at higher temperatures to be of value in diagnosing the structure of the bilayers. Thus by choice of a suitable spin label and a suitable temperature, this technique should prove useful for detection and diagnosis of lipid bilayer structure with a good degree of reliability. Caution must, of course be exercised, as with any spectroscopic technique. Spin labels will also be invaluable for more detailed studies of known interdigitated bilayers, which would be time- and material-consuming, if carried out using X-ray diffraction solely.

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.

Interaction of myelin basic protein with different ionization states of phosphatidic acid and phosphatidylserine.

Myelin basic protein (BP) has a perturbing effect on some lipids, causing, among other effects, a decrease in the temperature and enthalpy of the phase transition. This is believed to be a result of penetration of some hydrophobic residues of the protein partway into the lipid bilayer. Variations in the perturbing effect of BP on different acidic lipids has been attributed to the ability of the lipids to participate in intermolecular hydrogen bonding which inhibits penetration of the protein. Participation in intermolecular hydrogen bonding depends on the ionization state of the lipid as well as the type of lipid. In order to further test the dependence of the degree of penetration of BP on the intermolecular hydrogen bonding properties of lipids, the effect of BP on the phase transition of lipids in different ionization states was studied using differential scanning calorimetry. Dipalmitoylphosphatidic acid (DPPA) and dimyristoylphosphatidylserine (DMPS) were studied at different pH-values from 4 to 9.5. The results were compared to data obtained earlier with phosphatidylglycerol (PG), which is in the same ionization state at pH-values above 4, in order to distinguish the effects of pH on the protein from effects on the lipids. The perturbing effect of BP on PG increases with increase in pH. This is probably a result of the increasing hydrophobicity of the protein as the histidines become deprotonated, which allows greater penetration of the protein into the bilayer. In contrast, the effect on DPPA was greatest at low pH, where the state of ionization of the lipid is less than 1 and protein binding utilizes all of the hydrogen bond accepting sites (P-O-) on the lipid. BP had no perturbing effect on DPPA at higher pH where the state of ionization is between 1 and 1.5, and hydrogen bond accepting and donating sites (P-OH) are still available even after binding of the protein. Thus hydrogen bonding occurs at high pH and penetration of hydrophobic residues of the protein into DPPA is inhibited. BP had a large perturbing effect on DMPS at all pH values above 4 suggesting that lipid intermolecular hydrogen bonding does not occur in the presence of the protein and its hydrophobic residues consequently can penetrate into the bilayer. The protein may inhibit hydrogen bonding by binding electrostatically to the anionic hydrogen bond accepting group of PS.

Phase transitions and fatty acid spin label behavior in interdigitated lipid phases induced by glycerol and polymyxin.

Glycerol and polymyxin have been shown by X-ray diffraction to induce interdigitated bilayers in phosphatidylcholine (PC) and phosphatidylglycerol (PG), respectively (McDaniel, R.V., et al. (1983) Biochim. Biophys. Acta 731, 97-108; Ranck, J.-L. and Tocanne, J.-F. (1982) FEBS Lett. 143, 175-178). In the present study we have investigated the phase behavior of PC and PG in the presence of glycerol and polymyxin by differential scanning calorimetry and the use of fatty acid spin labels. Interdigitation causes a large increase in the order parameter of a fatty acid spin labeled near the terminal methyl, 16-doxylstearate, so that it was similar to that of a fatty acid labeled much closer to the polar head group region, 5-doxylstearate. Thus interdigitation abolishes the fluidity gradient found in a non-interdigitated bilayer. 16-Doxylstearate may be useful in detecting interdigitation of lipid bilayers caused by other substances. The different samples all went through two transitions on heating or cooling, or both. However, use of the fatty acid spin label showed that the molecular events during these transitions varies for different samples. The results suggested that PC-glycerol freezes from the liquid-crystalline phase into a non-interdigitated gel phase. This subsequently becomes interdigitated upon lowering the temperature a few degrees, in a low enthalpy transition. PG-polymyxin shows a similar behavior except that the enthalpy of the non-interdigitated gel to interdigitated phase transition is greater and the transition is reversible on heating. Thus on heating PG-polymyxin first goes through a transition from the interdigitated phase to a non-interdigitated gel phase and then, in a separate transition, to the liquid-crystalline phase. This occurs because the fatty acid chains in the presence of polymyxin become too disordered with increase in temperature to maintain the interdigitated state. PG-glycerol goes into the interdigitated state less readily than the other mixtures. If cooled rapidly, PG-glycerol freezes into a metastable phase which is more disordered than the interdigitated phase. It goes into the interdigitated phase in an exothermic transition on heating. An increase in fatty acid chain length causes greater steric hindrance to interdigitation but also increases the stabilizing energy gained by interdigitation.

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)

Changes in the composition of two molecular species of ethanolamine plasmalogen in normal human myelin during development.

Changes in the ratio of two molecular species of ethanolamine plasmalogen, PI-LE-1 and PI-PE-2, of human central nervous system myelin during development were measured by a TLC procedure. The ratio was found to decrease sharply with age up to 6 months as a result of an increase in the amount of PI-PE-2, believed to be a unique myelin lipid with 18:1 chains in both positions of the glycerol. The ratio continued to decrease gradually with age and did not reach the adult level until an age of 17 years.

Application of prostaglandin-profiling techniques to study the release of prostaglandins from the fetal lamb ductus arteriosus.

Through gas chromatographic techniques with capillary columns and electron-capture detection which allow the resolution of prostaglandins F2 alpha, D2, E2, thromboxane B2 and 6-ketoprostaglandin F1 alpha and their 15-keto-and 15-keto-13,14-dihydro metabolites, we have studied the release of these products from the ductus arteriosus in vitro. 6-Ketoprostaglandin F1 alpha was the major product in the incubation fluid while 15-keto-13,14-dihydro prostaglandin F2 alpha was the major product in the tissue. Prostaglandin E2 was almost undetectable in the fluid and tissue. Prostaglandin I2 formed in major proportions by the tissue is released mostly as 6-ketoprostaglandin F1 alpha, although minor amounts of its 15-keto-13,14-dihydro metabolites were detected. These results show differential release of prostaglandin types by this tissue, demonstrating formation and metabolism of endogenous prostaglandins at the same time.