A retrospective review of pregnancies on biologics for the treatment of dermatological conditions.

Psoriasis often first presents in young adulthood, with the average age of diagnosis in women being 28 years, thus in the prime reproductive years. In addition, approximately 50% of pregnancies worldwide are unplanned. Although biologic therapies have revolutionized the treatment of moderate-to-severe psoriasis, there are no controlled studies of biologics in pregnant women. The increasing use of these agents in women of childbearing age highlights the need to further assess their safety during pregnancy. Postmarketing experience regarding the safety of these drugs is accumulating and being published, with largely reassuring results. We present our real-world experience of 17 pregnancies occurring in women on treatment with biologic agents for dermatological conditions to further add to the body of knowledge.

Automated symptom and treatment side effect monitoring for improved quality of life among adults with diabetic peripheral neuropathy in primary care: a pragmatic, cluster, randomized, controlled trial.

AIMS: To evaluate the effectiveness of automated symptom and side effect monitoring on quality of life among individuals with symptomatic diabetic peripheral neuropathy. METHODS: We conducted a pragmatic, cluster randomized controlled trial (July 2014 to July 2016) within a large healthcare system. We randomized 1834 primary care physicians and prospectively recruited from their lists 1270 individuals with neuropathy who were newly prescribed medications for their symptoms. Intervention participants received automated telephone-based symptom and side effect monitoring with physician feedback over 6 months. The control group received usual care plus three non-interactive diabetes educational calls. Our primary outcomes were quality of life (EQ-5D) and select symptoms (e.g. pain) measured 4-8 weeks after starting medication and again 8 months after baseline. Process outcomes included receiving a clinically effective dose and communication between individuals with neuropathy and their primary care provider over 12 months. Interviewers collecting outcome data were blinded to intervention assignment. RESULTS: Some 1252 participants completed the baseline measures [mean age (sd): 67 (11.7), 53% female, 57% white, 8% Asian, 13% black, 20% Hispanic]. In total, 1179 participants (93%) completed follow-up (619 control, 560 intervention). Quality of life scores (intervention: 0.658 ± 0.094; control: 0.653 ± 0.092) and symptom severity were similar at baseline. The intervention had no effect on primary [EQ-5D: -0.002 (95% CI -0.01, 0.01), P = 0.623; pain: 0.295 (-0.75, 1.34), P = 0.579; sleep disruption: 0.342 (-0.18, 0.86), P = 0.196; lower extremity functioning: -0.079 (-1.27, 1.11), P = 0.896; depression: -0.462 (-1.24, 0.32); P = 0.247] or process outcomes. CONCLUSIONS: Automated telephone monitoring and feedback alone were not effective at improving quality of life or symptoms for people with symptomatic diabetic peripheral neuropathy. TRIAL REGISTRATION: ClinicalTrials.gov (NCT02056431).

Demyelination during anti-tumour necrosis factor therapy for psoriasis.

Anti-tumour necrosis factor (anti-TNF) therapies have been associated with neurological complications, including in rare cases demyelinating disease. It is currently unknown whether patients who have received more than one immunosuppressive agent or anti-TNF have a greater risk of demyelination. We report the case of a 37-year-old woman with psoriasis who presented with an acute episode of demyelination while on anti-TNF therapy. This case was complicated by the fact that progressive multifocal leukoencephalopathy was considered the likely diagnosis initially and was only definitively excluded by brain biopsy. This case demonstrates the difficulty establishing the correct diagnosis in patients with atypical presentations on immunomodulating therapies. We present this rare case of demyelination in a patient who received multiple immunosuppressive therapies to highlight this challenging clinical situation and discuss management with a literature review.

Genetic polymorphism in Methylenetetrahydrofolate Reductase chloride transport protein 6 (MTHFR CLCN6) gene is associated with keratinocyte skin cancer in a cohort of renal transplant recipients.

Background: Renal transplant recipients (RTRs) are at increased risk of keratinocyte cancer (KC), especially cutaneous squamous cell carcinoma (cSCC). Previous studies identified a genetic variant of the Methylenetetrahydrofolate Reductase (MTHFR) gene, C677T, which conferred a risk for diagnosis of cSCC in Irish RTRs. Objective: We sought to find further genetic variation in MTHFR and overlap genes that may be associated with a diagnosis of KC in RTRs. Methods: Genotyping of a combined RTR population (n = 821) from two centres, Ireland (n = 546) and the USA (n = 275), was performed. This included 290 RTRs with KC and 444 without. Eleven single nucleotide polymorphisms (SNPs) in the MTHFR gene and seven in the overlap gene MTHFR Chloride transport protein 6 (CLCN6) were evaluated and association explored by time to event analysis (from transplant to first KC) using Cox proportional hazards model. Results: Polymorphism at MTHFR CLCN6 (rs9651118) was significantly associated with KC in RTRs (HR 1.50, 95% CI 1.17-1.91, p < 0.00061) and cSCC (HR 1.63, 95% CI 1.14-2.34, p = 0.007). A separate SNP, MTHFR C677T, was also significantly associated with KC in the Irish population (HR 1.31, 95% CI 1.05-1.63, p = 0.016), but not American RTRs. Conclusions: We report the association of a SNP in the MTHFR overlap gene, CLCN6 and KC in a combined RTR population. While the exact function of CLCN6 is not known, it is proposed to be involved in folate availability. Future applications could include incorporation in a polygenic risk score for KC in RTRs to help identify those at increased risk beyond traditional risk factor assessment.

Critical and off-critical miscibility transitions in model extracellular and cytoplasmic myelin lipid monolayers.

Monolayers based on the composition of the cytoplasmic (CYT) or extracellular (EXT) sides of the myelin bilayer form coexisting immiscible liquid phases similar to the liquid-ordered/liquid-disordered phases in phospholipid/cholesterol monolayers. Increasing the temperature or surface pressure causes the two liquid phases to mix, although in significantly different fashion for the CYT and EXT monolayers. The cerebroside-rich EXT monolayer is near a critical composition and the domains undergo coalescence and a circle-to-stripe transition along with significant roughening of the domain boundaries before mixing. The phase transition in the cerebroside-free cytoplasmic side occurs abruptly without domain coalescence; hence, the cytoplasmic monolayer is not near a critical composition, although the domains exhibit shape instabilities within 1-2 mN/m of the transition. The change in mixing pressure decreases significantly with temperature for the EXT monolayer, with dΠ(crit)/dT ∼ 1.5 mN/m/°C, but the mixing pressure of the CYT monolayer varies little with temperature. This is due to the differences in the nonideality of cholesterol interactions with cerebrosides (EXT) relative to phospholipids (CYT). EXT monolayers based on the composition of white matter from marmosets with experimental allergic encephalomyelitis (EAE), an animal model of multiple sclerosis, remain phase-separated at higher surface pressures than control, while EAE CYT monolayers are similar to control. Myelin basic protein, when added to the CYT monolayer, increases lipid miscibility in CYT monolayers; likely done by altering the dipole density difference between the two phases.

Lipid intermolecular hydrogen bonding: influence on structural organization and membrane function.

The great variety of different lipids in membranes, with modifications to the hydrocarbon chains, polar groups and backbone structure suggests that many of these lipids may have unique roles in membrane structure and function. Acidic groups on lipids are clearly important, since they allow interaction with basic groups on proteins and with divalent cations. Another important property of certain lipids is their ability to interact intermolecularly with other lipids via hydrogen bonds. This interaction occurs through acidic and basic moieties in the polar head groups of phospholipids, and the amide moiety and hydroxyl groups on the acyl chain, sphingosine base and sugar groups of sphingo- and glycolipids. The putative ability of different classes of lipids to interact by intermolecular hydrogen bonding, the molecular groups which may participate and the effect of these interactions on some of their physical properties are summarized in Table IX. It is frequently questioned whether intermolecular hydrogen bonding could occur between lipids in the presence of water. Correlations of their properties with their molecular structures, however, suggest that it can. Participation in intermolecular hydrogen bonding increases the lipid phase transition temperature by approx. 8-16 Cdeg relative to the electrostatically shielded state and by 20-30 Cdeg relative to the repulsively charged state, while having variable effects on the enthalpy. It increases the packing density in monolayers, possibly also in the liquid-crystalline phase in bilayers, and decreases the lipid hydration. These effects can probably be accounted for by transient, fluctuating hydrogen bonds involving only a small percentage of the lipid at any one time. Thus, rotational and lateral diffusion of the lipids may take place but at a slower rate, and the lateral expansion is limited. Intermolecular hydrogen bonding between lipids in bilayers may be significantly stabilized, despite the presence of water, by the fact that the lipids are already intermolecularly associated as a result of the hydrophobic effect and the Van der Waals' interactions between their chains. The tendency of certain lipids to self-associate, their asymmetric distribution in SUVs, their preferential association with cholesterol in non-cocrystallizing mixtures, their temperature-induced transitions to the hexagonal phase and their inhibitory effect on penetration of hydrophobic residues of proteins partway into the bilayer can all be explained by their participation in intermolecular hydrogen bonding interactions.(ABSTRACT TRUNCATED AT 400 WORDS)

Calorimetric and fatty acid spin label study of subgel and interdigitated gel phases formed by asymmetric phosphatidylcholines.

Several saturated asymmetric and symmetric phosphatidylcholines were studied by ESR spectroscopy and differential scanning calorimetry in order to determine the behavior of a fatty acid spin labeled near its terminal methyl, 16-doxylstearate, in the mixed interdigitated gel phase and the Lc subgel phase and other properties of these lipids. This spin label was motionally restricted in the mixed interdigitated gel phases of 18:10PC and 18:12PC. The motional restriction was similar to that reported earlier for fully interdigitated phases. This spin label was motionally restricted almost to the same degree in 10:18PC suggesting that this asymmetric lipid may also form a mixed interdigitated bilayer. In contrast the spin label had more motion in the gel phase of 18:14PC than in symmetric forms of PC, consistent with conclusions from X-ray diffraction studies that this less asymmetric lipid does not form a mixed interdigitated phase. The spin label was partially frozen out of the Lc subgel phases of symmetric forms of PC and 18:14PC formed by storage at low temperature. The phase behavior of the other asymmetric lipids also depended on the sample history. Storage at low temperature caused 10:18PC and 18:12PC to go into ordered phases. The enthalpy of the transition of these ordered phases to the liquid-crystalline phase was 2-2.4-times greater than that of the transition of the gel phase formed on cooling back from the liquid-crystalline phase. The temperature of this high enthalpy transition was 0.8 K below that of the lower enthalpy gel to liquid-crystalline phase transition for 18:12PC, but 4.6 K higher for 10:18PC. The spin label was frozen out of these ordered phases, as it was out of the Lc subgel phases, suggesting that 18:12PC and 10:18PC may also form an Lc phase. 18:10PC was not observed to form an ordered phase although storage of the sample at low temperatures did affect the temperature of its transition from the liquid-crystalline phase back to the gel phase upon cycling through its phase transition.

Interdigitated gel phase bilayers formed by unsaturated synthetic and bacterial glycerolipids in the presence of polymyxin B and glycerol.

The ability of synthetic phosphoglycerolipids with a cis mono-unsaturated acyl chain in the 2-position and a saturated chain in the 1-position of glycerol to form interdigitated gel phase bilayers in the presence of amphipathic substances was monitored using a fatty acid spin label, 16-doxylstearic acid, and a phosphatidylglycerol spin label containing 16-doxylstearic acid. These spin labels become significantly more motionally restricted in an interdigitated gel phase bilayer than in a non-interdigitated gel phase bilayer. The results indicated that polymyxin B and polymyxin B nonapeptide caused interdigitation of 1-palmitoyl,2-oleoyl-phosphatidylglycerol (POPG) and glycerol caused interdigitation of 1-stearoyl,2-oleoyl-phosphatidylcholine (SOPC), similar to their effects on disaturated lipids. The fluidity gradient present in non-interdigitated gel phase bilayers was abolished. However, glycerol did not cause POPG to become interdigitated, in contrast to SOPC. We reported earlier that there is a kinetic barrier to interdigitation of saturated PG in the presence of glycerol, in contrast to saturated PC. This barrier is even greater for the unsaturated species of PG. Furthermore, these compounds lowered the gel to liquid-crystalline phase transition temperatures of the unsaturated lipids more than of saturated lipids suggesting that the interdigitated bilayer of the former may be less ordered or less stable than that of the latter. Since polymyxin B is an antibiotic we also examined its effect on a lipid extract from the Gram-negative bacteria Pseudomonas aeruginosa in order to assess whether interdigitation might be involved in its mechanism of bactericidal or bacteriostatic effect. Polymyxin B and polymyxin B nonapeptide also caused motional restriction of a small percentage (about 13% at -2 degrees C and 25% at -14 degrees C for polymyxin B) of the spin label in the lipid extract at low temperatures, where the lipid is in the gel phase, consistent with formation of a small domain of interdigitated bilayer lipid. However, the degree of immobilization was less than that in the interdigitated bilayers of the synthetic unsaturated lipids. This may be a result of the heterogeneous nature of the lipids in the extract. However, it cannot be ruled out that the motional restriction of the spin label in this extract may be caused by something other than interdigitation. Thus the results with the lipid extract are less conclusive of interdigitation than for the synthetic lipids. A motionally restricted population was not detectable at higher temperatures.(ABSTRACT TRUNCATED AT 400 WORDS)

A transmembrane potential does not affect the vertical location of charged lipid spin labels with respect to the surface of a phosphatidylcholine bilayer.

The effect of a transmembrane potential on the vertical location of a charged lipid in a neutral phosphatidylcholine (PC) lipid bilayer has been investigated using negatively and positively charged spin-labeled lipids. A transmembrane potential was generated across extruded large unilamellar vesicles either by using a K+/Na+ ion gradient and a K+ ionophore or by using a pH gradient. Since a transmembrane potential could have opposing effects on lipids in the inner and outer monolayer, some of the acidic spin labels were asymmetrically located in the inner monolayer as a result of a pH gradient. No significant effect on their order parameters was observed upon applying a transmembrane potential. The internal dipole potential of the bilayer was modified by using dialkyl-PC or by incorporating 10 mol% phloretin, or 6-ketocholestanol in the PC, but a transmembrane potential still had no detectable effect on the spin labeled lipids. Therefore, it is concluded that the electrochemical potential across membranes probably does not cause a significant change in the vertical location of charged lipids with respect to the surface of a PC bilayer. This suggests that polar interactions and/or van der Waals interactions between the spin probe and the surrounding lipids stabilize the overall structure of the membranes and these interactions are not disrupted by a selective effect of the transmembrane potential on the charged lipids.

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.

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.

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.

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.

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.

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.

Use of spin labels to determine the percentage of interdigitated lipid in complexes with polymyxin B and polymyxin B nonapeptide.

Long chain spin labels with the nitroxide group located near the terminal methyl of the chain were used to determine the percentage interdigitated lipid in complexes of polymyxin B (PMB) and polymyxin B nonapeptide (PMBN) with the acidic lipids dipalmitoylphosphatidylglycerol (DPPG) and dipalmitoylphosphatidic acid (DPPA) at varying mole ratios of drug to lipid and at different pH values. These spin labels are more motionally restricted in the interdigitated than in the non-interdigitated gel phase bilayer. This allows determination of the percentage interdigitated lipid by resolution of the spectrum into motionally restricted and more mobile components. At nonsaturating concentrations of PMB, significantly more DPPG than that which can be maximally PMB-bound, becomes interdigitated. As the temperature approaches the gel to liquid crystalline phase transition temperature, the bilayer becomes progressively non-interdigitated. The ESR spectrum indicates that PMB also causes interdigitation of DPPA. However, in contrast to DPPG, the amount of DPPA which is interdigitated at pH 6, is less than the amount which is expected to be PMB-bound. This is attributed to the ability of DPPA to participate in lateral interlipid hydrogen bonding interactions. Such lateral interactions would be abolished in the interdigitated bilayer and thus they are expected to inhibit its formation. At pH 9, where the interlipid interactions of DPPA are weakened, PMB induces even more lipid than that which is PMB-bound to become interdigitated. Indeed, the percentage interdigitated lipid is even greater than found for DPPG. This may be partly a result of the greater negative charge of DPPA at this pH. A greater repulsive negative charge is expected to favor interdigitation. PMBN is less effective than PMB at inducing interdigitation of DPPG and causes little or no interdigitation of DPPA at pH 6, even at saturating concentrations. PMBN also does not lower the phase transition temperature of DPPA at pH 6 as much as PMB. At pH 9, the effect of PMBN on DPPA is more similar to the effect of PMB. However, even for DPPG, and DPPA at pH 9, PMBN does not maintain interdigitation of the lipids at higher temperatures as effectively as PMB. PMBN's smaller perturbing effect and greatly decreased ability to cause interdigitation of DPPA at pH values below 9 may be related to a decreased ability to cause lateral separation of the lipid molecules, which is necessary in order to weaken the interlipid interactions.(ABSTRACT TRUNCATED AT 400 WORDS)

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.

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.