Liposomes as Immunoadjuvants and Vaccine Carriers: Antigen Entrapment

Successful use of liposomes as immunological adjuvants in vaccines requires simple, easy to scale up technology capable of high-yield antigen entrapment. Recent work from this laboratory has led to the development of techniques that can generate liposomes of various sizes containing soluble antigens such as proteins or particulate antigens such as whole, live, or attenuated bacteria or viruses. Entrapment of proteins is carried out by the dehydration-rehydration procedure, which entails freeze-drying of a mixture of "empty" small unilamellar vesicles and free antigens. Upon rehydration, the large multilamellar vesicles that are formed incorporate up to 80% of the antigen used. When such liposomes are microfluidized in the presence of nonentrapped material, their size is reduced to about 100 nm in diameter, with much of the originally entrapped antigen still associated with the vesicles. A similar technique applied to the entrapment of particulate antigens (e.g., Bacillus subtilis spores) consists of freeze-drying giant vesicles (4-5 μm in diameter) in the presence of spores. On rehydration and sucrose gradient fractionation of the suspension, up to 27% of the spores used are associated with generated giant liposomes of similar mean size.     

Vaccine entrapment in liposomes.

The use of liposomes as carriers of peptide, protein, and DNA vaccines requires simple, easy-to-scale-up technology capable of high-yield vaccine entrapment. Work from this laboratory has led to the development of techniques that can generate liposomes of various sizes, containing soluble antigens such as proteins and particulate antigens (e.g., killed or attenuated bacteria or viruses), as well as antigen-encoding DNA vaccines. Entrapment of vaccines is carried out by the dehydration-rehydration procedure which entails freeze-drying of a mixture of "empty" small unilamellar vesicles and free vaccines. On rehydration, the large multilamellar vesicles formed incorporate up to 90% or more of the vaccine used. When such liposomes are microfluidized in the presence of nonentrapped material, their size is reduced to about 100 nm in diameter, with much of the originally entrapped vaccine still associated with the vesicles. A similar technique applied for the entrapment of particulate antigens (e.g., Bacillus subtilis spores) consists of freeze-drying giant vesicles (4-5 microm in diameter) in the presence of spores. On rehydration and sucrose gradient fractionation of the suspension, up to 30% or more of the spores used are associated with generated giant liposomes of similar mean size.     

Ionophoric properties of the proteolipid apoprotein from bovine brain myelin

Ionophoric properties of the Proteolipid Apoprotein have been assayed. This is a highly purified and delipidated intrinsic myelin membrane protein, isolated from bovine brain white matter. The preparation of myelin membrane vesicles or the incorporation of purified protein into Dimiristoylphosphatidylcholine liposomes have been carried out. According to our results, the myelin Proteolipid protein may act as a Na+ and Rb+ (K+) unidirectional ionophoretic channel, which main physiological role could be related to the maintenance of ionic equilibrium of myelin sheath around the axons.     

Poster Sessions BP03: Myelin,Demyelination and Diseases of Myelin. Myelin and myelination in PLP‐null mice

The myelin proteolipid protein (PLP) is the major structural protein of CNS myelin, accounting for approximately half of total myelin protein. We studied synthesis and accumulation of myelin components for two months postnatally in PLP‐null mice and age‐matched controls. Accumulation of myelin, as assayed by levels of whole brain cerebroside and myelin basic protein, was normal in the knockout mice. The rate of cerebroside synthesis in the knockout mice was also normal. Myelin was isolated at several ages during development, using a standard subcellular fractionation protocol. The yield of ‘purified myelin’ isolated from a large particle (crude mitochondrial) fraction was reduced in PLP‐null mice, but increased amounts of ‘myelin’ were obtained in the small particle (crude microsomal) fraction. This ‘myelin’ in the crude microsomal fraction was identified as such by flotation on 0.85 m sucrose and the myelin‐characteristic 2 : 1 molar ratio of cholesterol to cerebroside. This suggests myelin from PLP‐null mice is physically more fragile than normal myelin, and that during tissue dispersion, much more PLP‐null myelin is fragmented into small vesicles than is the case for normal myelin. Three hours after intracranial injection of tritiated acetate into PLP‐null mice, cerebroside in myelin isolated from the large particle fraction was at a similar specific radioactivity to that isolated from the small particle (crude microsomal) fraction, suggesting that the most recently deposited PLP‐null myelin is not preferentially unstable. The increased fragility evident during tissue dispersion is indicative of an underlying structural abnormality in PLP‐null myelin. Whether this inherent structural instability affects myelin metabolism is under investigation. Acknowledgements: Supported by USPHS & NMSS grants.     

Giant liposomes in physiological buffer using electroformation in a flow chamber

We describe a method to obtain giant liposomes (diameter 10-100 μm) in solutions of high ionic strength to perform a membrane-binding assay under physiological conditions. Using electroformation on ITO electrodes, we formed surface-attached giant liposomes in solutions of glycerol in a flow chamber and then introduced solutions of high ionic strength (up to 2 M KCl) into this chamber. The ionic solution exchanged with the isoosmolar glycerol solution inside and outside the liposomes. An initial mismatch in index of refraction between the inside and outside of liposomes allowed for the observation of solution replacement. Ions and small polar molecules exchanged into and out of surface-attached liposomes within minutes. In contrast, liposomes formed in solutions of macromolecules retained molecules larger than 4 kDa, allowing for encapsulation of these molecules for hours or days even if the solution outside the liposomes was exchanged. We propose that solutes entered liposomes through lipid tubules that attach liposomes to the film of lipids on the surface of the ITO electrode. The method presented here makes it straightforward to perform flow-through binding assays on giant liposomes under conditions of physiological ionic strength. We performed a membrane-binding assay for annexin V, a calcium-dependent protein that binds to phosphatidylserine (PS). The binding of annexin V depended on the concentration of PS and decreased as ionic strength increased to physiological levels.     

Preparation and some properties of giant liposomes and proteoliposomes

Optimal conditions for formation of giant liposomes and proteoliposomes were investigated. A suspension of small unilamellar vesicles made of various phospholipids in a buffer of 0-3 M KCl, 0.1 mM EDTA, and 20 mM MOPS (pH 7.0) was subjected to a freeze-thaw treatment. Giant multilamellar liposomes of diameter ranging from 10 to 60 microns were found to form from phospholipid mixtures containing phosphatidylethanolamine as a major component and phosphatidylserine as a minor component. The concentration of KCl optimal for the giant vesicle formation was 30-500 mM. By applying a patch-pipette to a giant liposome, suitable conditions for obtaining a high-resistance (giga-ohm) seal were sought. It was found that use of a patch-pipette of relatively small tip diameter (less than 1 micron), the presence of divalent metal cations in the suspension medium and inflation of vesicles in a hypotonic solution facilitated giga-seal formation. In a suspension of asolectin (soybean phospholipid) vesicles which had been subjected to the freeze-thaw treatment, giant unilamellar vesicles were found. They could be held on the tip of a suction pipette and impaled with a microelectrode filled with an EGTA solution. Small unilamellar proteoliposomes were prepared by the cholate-dialysis method from asolectin and sarcoplasmic reticulum vesicles, and were subjected to a freeze-thaw cycle. When the ratio of exogenous phospholipid to protein was larger than 10, giant multilamellar vesicles were formed.(ABSTRACT TRUNCATED AT 250 WORDS)     

Encapsulation of Chicken Egg Yolk Immunoglobulin G (IgY) by Liposomes

Encapsulation of antibodies isolated from chicken egg yolk (IgY) in egg lecithin/cholesterol liposomes was attempted. IgY was successfully encapsulated into the liposomes by using the dehydration-rehydration method. Electron microscopic observation demonstrated that the liposomes prepared by this method were large multilamellar vesicles with a diameter of several μm. The encapsulation efficiency was improved by increasing the rehydration temperature to 60°C. The cholesterol/lecithin ratio also affected the efficiency, giving the highest value at a ratio of 1/4 (mol/mol). Some efflux of glucose through the liposomal membranes was observed, particularly for the liposome with a low cholesterol content, but that of IgY was not detected, irrespective of the cholesterol content. Encapsulation reduced the activity loss of the IgY antibodies under acidic conditions. IgY encapsulated in the liposomes was also markedly resistant to pepsin hydrolysis, which usually results in complete loss of activity with unencapsulated IgY, suggesting that liposomal encapsulation is an effective means for protecting IgY under gastric conditions.     

Electrical activity and structural correlates of giant nerve fibers in Kuruma shrimp (Penaeus japonicus)

Morphology and recordings of electrical activity of Kuruma shrimp (Penaeus japonicus) giant medullated nerve fibers were carried out. A pair of giant fibers with external diameter of about 120 μ and 10 μ in myelin thickness were found in the ventral nerve cord. The diameter of the axon is about 10 μ. Thus there is a wide gap between the axon and the external myelin sheath. Each axon is doubly coated directly by Schwann cells and indirectly by the myelin sheath layer which is produced by those Schwann cells. Impulse conduction velocities of these giant fibers showed a range between 90–210 m/sec at about 22°C. Large action potentials (up to 113 mV, rise time of 0.16–0.3 msec, maximum rate of rise of 650–1250 V/sec, half decay time of 0.2–0.3 msec, maximum rate of fall of 250–450 V/sec and total duration of less than 1.5 msec) could be obtained by inserting microelectrodes or by longitudinal insertion of 25 μ diameter capillary electrodes into the gap but no DC-potential difference was observed across the myelin sheath. Transmyelin electrical parameters were very favorable for fast impulse conduction: myelin resistance of 3 × 10⁴ Ω cm²; time constant of 0.38 msec; myelin capacitance of 1.35 × 10^?8 F/cm²; gap fluid resistivity of 23 Ω cm. The existence of nodes of Ranvier could not be demonstrated morphologically, but electrophysiological evidence suggests that a type of saltatory conduction occurs in these giant fibers.     

An electrophoretic analysis of proteolipids from different rat brain subcellular fractions

Proteolipid proteins were extracted from adult rat brain subcellular fractions and purified by chromatography on Sephadex LH-60. Polyacrylamide gel electrophoresis of the delipidized proteins, in the presence or absence of 8 M urea, was carried out with all fractions. The distribution of the various types of proteolipid proteins was studied and their molecular weight calculated by the Ferguson relationship. Several bands of proteolipid proteins were found in the five membrane fractions analyzed. Some of them, such as the 17.5 K and 37 K components were very prominent in mitochondria and synaptosomes. The 30 K component was found in myelin-derived membranes and in microsomes, while the 20 K and 25 K proteolipid proteins were present in all subcellular fractions. The 30 K component (proteolipid protein (PLP)), typical of the purified myelin membranes, showed a similar distribution to that of 2′,3′-cyclic-nucleotide 3′-phosphohydrolase (EC 3.1.4.37) activity, while the other major proteolipid protein present in all subcellular fractions (25 K) did not show such parallelism, indicating that it might not be an exclusive component of myelin. The electrophoretic pattern of microsomal proteolipid proteins did not show the high molecular weight components (aggregates of PLP) which are found in myelin. Furthermore, the 30 K component showed a smaller $\text{Y}{}_0$ value than that of the 30 K found in myelin. Thus the presence of 30 K proteolipid protein in microsomes should not be considered as being due to myelin contamination.     

Spore morphology of Isoëtes (Isoëtaceae) from China

Micromorphological characters of mega- and microspores of five Isoëtes species from China were observed under scanning electron microscope (SEM). Megaspores of I. hypsophila have a levigate ornamentation, and are 358 µm in diameter (mean, n=30), microspores of this species have a gyrus ornamentation, and are 22 µm in length (mean, n=30); megaspores of I. yunguiensis have a cristate-reticulate ornamentation, with a size of 390 µm in diameter (mean, n=30), while microspores nearly smooth with some fine granulates, and with a size of 22 µm in length (mean, n=30); megaspores of I. taiwanensis are tuberculate and 312 µm in diameter (mean, n=30), echinate microspores are 24 µm in length (mean, n=30); cristate megaspores of I. sinensis are 409 µm in diameter (mean, n=30), and echinate microspores are 28 µm in length (mean, n=30); megaspores of I. orientalis have a cristate-reticulate ornamentation, with diameter of 420 µm (mean, n=30), microspores of this species have an echinate-tuberculate ornamentation with a size of 34 µm in length (mean, n=30). Based on the morphological characters of spores and chromosome numbers among taxa, it was found that mean spore size in each species of Isoëtes generally reflected the ploidy level. I. hypsophila, I. yunguiensis and I. taiwanensis, with smaller mean spore size, are diploidy with 2n=22; I. sinensis (2n=44) and I. orientalis (2n=66), with larger mean spore size, are polyploidy. The spore morphology of Isoëtes still plays an important role in identification and classification of Isoëtes from China.     

Chamaebotrys prolifera sp. nov. (Rhodymeniaceae, Rhodophyta) from Puerto Rico, Caribbean Sea, based on morphology and small subunit rDNA sequences

A new rhodymeniacean species, Chamaebotrys prolifera , is described from a shallow water habitat in Puerto Rico, representing the first occurrence of the genus in the Atlantic Ocean. Plants, to 5 cm across, are decumbent and comprised of compressed vesicles that are originally proliferously branched at the perimeter. Older vesicles become branched from their dorsal surfaces as well. Branches are septate at their origin and become irregularly shaped with age. Anastomoses between adjacent vesicles is common. Individual vesicles measure to 15 mm in broadest dimension. Vesicle walls consist of two layers of medullary cells and two layers of cortical cells. Tetrasporangia, which occur in diffuse nemathecia, are spherical, to 30 µm in diameter and are cut off terminally from an inner cortical cell. Cystocarps are hemispherical measuring to 800 µm in diameter and 350 µm in height. Spermatangia are apparently cut off randomly from outer cortical cells across the thallus surface. Molecular evidence confirms placement of Chamaebotrys within the Rhodymeniaceae.     

Giant liposomes: a model system in which to obtain patch-clamp recordings of ionic channels.

Cell-size, giant liposomes have been formed by submitting a mixture of asolectin lipid vesicles and native membranes from Torpedo, highly enriched in acetylcholine receptor (AcChR), to a partial dehydration/rehydration cycle [Criado, M., & Keller, B. U. (1987) FEBS Lett. 224, 172-176]. Giant liposomes can be prepared in bulk quantities, in the absence of potentially damaging detergents or organic solvents, and their formation is mediated by membrane fusion phenomena. In fact, fluorescence microscopy and freeze-fracture data indicate that protein and lipid components of the initial membranes and lipid vesicles are homogenously distributed in the resulting liposomes. Giant liposomes containing AcChR have been used as a model to evaluate whether this system can be used to monitor the activity of ionic channels by using high-resolution, patch-clamp techniques. Excised liposome patches in an "inside-out" configuration have been used in this work. We find that the most frequent pattern of electrical activity in response to the presence of acetylcholine in the patch pipet corresponds to a cation-specific channel exhibiting a dominant conductance level and a sublevel of approximately 78 and 25 pS, respectively. Such channel activity exhibits the pharmacological specificity, ion channel activation, ion selectivity, and desensitization properties expected from native Torpedo AcChR. Thus, it appears that the giant liposome technique offers a distinct advantage over other reconstitution procedures in that it provides a unique opportunity to undertake simultaneous biochemical, morphological, and electrophysiological studies of the incorporated ionic channel proteins.     

Giant liposomes in physiological buffer using electroformation in a flow chamber

We describe a method to obtain giant liposomes (diameter 10-100 microm) in solutions of high ionic strength to perform a membrane-binding assay under physiological conditions. Using electroformation on ITO electrodes, we formed surface-attached giant liposomes in solutions of glycerol in a flow chamber and then introduced solutions of high ionic strength (up to 2 M KCl) into this chamber. The ionic solution exchanged with the isoosmolar glycerol solution inside and outside the liposomes. An initial mismatch in index of refraction between the inside and outside of liposomes allowed for the observation of solution replacement. Ions and small polar molecules exchanged into and out of surface-attached liposomes within minutes. In contrast, liposomes formed in solutions of macromolecules retained molecules larger than 4 kDa, allowing for encapsulation of these molecules for hours or days even if the solution outside the liposomes was exchanged. We propose that solutes entered liposomes through lipid tubules that attach liposomes to the film of lipids on the surface of the ITO electrode. The method presented here makes it straightforward to perform flow-through binding assays on giant liposomes under conditions of physiological ionic strength. We performed a membrane-binding assay for annexin V, a calcium-dependent protein that binds to phosphatidylserine (PS). The binding of annexin V depended on the concentration of PS and decreased as ionic strength increased to physiological levels.     

Brain Glutamate Decarboxylase: Properties of Its Calcium-Dependent Binding to Liposomes and Kinetics of the Bound and the Free Enzyme

Abstract: In the present work we describe several properties of the Ca²⁺-dependent binding of glutamate decarboxylase (GAD) to phosphatidylcholine-phosphatidylserine liposomes. The binding occurs very rapidly, is dependent on temperature in the range 23–37°C, is inhibited up to 35% by K⁺ in a concentration-dependent manner and is slightly increased when the dielectric constant of the medium is decreased by 3% ethanol. The association of GAD and liposomes is very firm, since EGTA displaces only 40% of the bound enzyme and Triton X-100 about 55%. Since apparently only part of the total GAD is able to bind to the liposomes and in a previous study two forms of GAD activity have been identified kinetically, we compared the activations by pyridoxal 5'-phosphate (PLP) of the soluble and the bound GAD, as well as their inhibition by PLP oxime- O -acetic acid. The bound GAD was activated 150–265% by 10⁻⁶ to 10⁻⁴ m -PLP, whereas the activation of GAD that remained soluble was only 65–110% in the same PLP concentration range. In the absence of PLP, the bound GAD was less inhibited by the PLP oxime- O -acetic acid than the soluble GAD, but the inhibition was similar when 0.1 m m -PLP was added. In contrast, activity of both the soluble and the bound GAD was totally blocked by aminooxyacetic acid. Endogenous PLP did not bind to liposomes under the experimental conditions inducing GAD binding. We conclude that the binding of GAD to negatively charged liposomes is primarily ionic. Furthermore, the GAD molecules that bind to the liposomes seem to be deficient in free PLP and therefore, are probably more susceptible to regulation by the coenzyme. These conclusions may be relevant to the hypothesis of a coupling between synthesis and release of GABA in inhibitory nerve endings.     

Size and Surface Charge Properties of Myelin Vesicles from Normal and Diseased (Multiple Sclerosis) Brain

Differences have been observed between myelin vesicles prepared from normal human central nervous system and from white matter of patients who died with multiple sclerosis (MS). The mean cross-sectional area of the vesicles was 5.69 +/- 0.17 micron 2 from normal myelin and 3.71 +/- 0.28 micron 2 for diseased myelin. Vesicle size was reduced to 4.08 +/- 0.21 micron 2 when normal myelin vesicles were prepared in the presence of 0.1 mM EDTA. The presence of Ca2+ during the preparation of the vesicles had no effect on the mean cross-sectional area. In the case of MS myelin vesicles, 0.1 mM EDTA had no effect on vesicle size, whereas the presence of Ca2+ increased the vesicle size from 3.71 +/- 0.28 to 5.40 +/- 0.31 micron 2. Electrokinetic analysis revealed that the electrophoretic mobility of normal myelin vesicles was -5.169 +/- 0.193 X 10(-8) compared with -6.093 +/- 0.202 X 10(-8) m2 s-1 V-1 for the MS myelin vesicles. The presence of 0.1 mM EDTA increased the electrophoretic mobility of the normal vesicles to -6.483 +/- 0.151 X 10(-8) m2 s-1 V-1 but did not significantly affect that of the MS vesicles. Addition of 0.1 mM Ca2+ decreased the electrophoretic mobility of both normal and MS vesicles to similar mobilities. From these data, the surface charge densities were calculated for both normal and MS myelin vesicles and found to be -2.93 and -5.39 mV m-1, respectively. The phase transition temperature determined by wide-angle x-ray diffraction studies was 63 degrees C for normal myelin vesicles and 43 degrees C for MS myelin vesicles.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of Rumpshaker Mutation on CNS Myelin Composition and Structure

Abstract: Myelinated CNS tissues from homozygous/hemizygous and heterozygous jimpy rumpshaker jp ^rsh mutant mice were examined to determine the consequences on myelin structure of this mutation in the proteolipid protein (PLP) gene. Polyacrylamide gel electrophoresis and immunoblotting of brain homogenates confirmed that there was a decrease in PLP levels on the B6C3 genetic background onto which this gene was bred. We also observed an increase in level of a protein band that could correspond to the uncharacterized 10-kDa PLP previously reported in jp ^rsh mice on an Rb(1.3) 1Bnr background. High-performance TLC and densitometry of lipids from brain homogenate and isolated myelin revealed a decrease in content of cerebrosides and sulfatides. Electron microscopy on optic nerves revealed that normal radial component is retained in jp ^rsh myelin, further substantiating that PLP is not a component of this junctional complex. X-raydiffraction measurements on unfixed optic nerves showed that the jp ^rsh period is 5–10 Å larger than normal. Moreover, jp ^rsh optic nerve myelin was unstable, as evidenced by a continual increase in the period postdissection. jp ^rsh myelin that was equilibrated at varying pH and ionic strength typically had a larger than normal period under all conditions (both swelling and compacting). Our findings thus demonstrate that the biochemical abnormalities in the jp ^rsh mutant correlate with a wider periodicity and less stable packing of the myelin.     

Inhibition of murine relapsing experimental autoimmune encephalomyelitis by immune tolerance to proteolipid protein and its encephalitogenic peptides.

Tolerization of SJL/J mice with splenocytes coupled with proteolipid protein (PLP), the major protein component of central nervous system myelin, resulted in dramatic inhibition of relapsing experimental autoimmune encephalomyelitis (R-EAE) induced by mouse spinal cord homogenate (MSCH). Mice tolerized with splenocytes coupled with MSCH (a complex mixture of neuroantigens) or with purified PLP, but not purified myelin basic protein, were resistant to the development of clinical and histologic R-EAE. In addition, mice rendered tolerant to an encephalitogenic peptide of PLP were significantly protected, whereas mice tolerized to a nonencephalitogenic peptide of PLP were highly susceptible, to the induction of MSCH-induced R-EAE. Thus, immune responses directed against encephalitogenic regions of PLP appear to play a major role in the development of R-EAE induced by MSCH in SJL/J mice. These results also indicate that determinant-specific immune tolerance is a feasible approach to the regulation of a disease that involves autoimmune responses to a variety of Ag.     

Myelin proteolipid protein is not esterified at the site of synthesis

Brain slices prepared from 20-day old rats were incubated with [³H]palmitic acid to study its incorporation into myelin proteins. After separation by SDS-PAGE, most of the label was found to be associated with the major proteolipid protein (PLP) and with the intermediate protein (I). The radioactivity measured in PLP at short incubation times was shown to be due to palmitic acid bound to the protein by ester linkages. Time-course incorporation of [³H]palmitic acid into PLP of fraction SN₄ (a myelin like membrane) and of purified myelin showed that the former was poorly labeled and no relationship of the type ‘precursor-product’ between these fractions could be detected. Incorporation of the fatty acid into PLP was not affected by inhibition of the synthesis or transport of myelin PLP with cycloheximide or colchicine, indicating that the pool of PLP that can be acylated must be larger than the extramyelin pool. Addition of unlabeled palmitic acid to the incubation medium, 30 min after the addition of [³H]palmitate, stopped the appearance of label in myelin PLP almost immediately, indicating that there is no significant extramyelin pool of PLP destined for transport into myelin. The results presented in this paper strongly suggest that esterification of PLP takes place in the myelin membrane or at a site very close to it.     

The Effect of Chronic Treatment with the GABA Transaminase Inhibitors γ-Vinyl-GABA and Ethanolamine-O-Sulphate on the In Vivo Release of GABA from Rat Hippocampus

Abstract: The effects of chronic treatment with the specific, mechanism-based, irreversible inhibitors of 4-aminobutyrate aminotransferase (EC 2.6.1.19; GABA transaminase), ethanolamine O -sulphate (EOS), and 4-aminohexenoate [vigabatrin; γ-vinyl-GABA (GVG)] on the extracellular concentrations of GABA in the hippocampus have been studied using in vivo microdialysis in conscious animals. Oral dosing [3 mg/ml of drinking water, giving doses of GVG of 194 ± 38 mg/kg/day and of EOS of 303 ± 42 mg/kg/day (mean ± SD)] was followed by microdialysis at 2, 8, and 21 days. The basal outflow of GABA (in the range of ∼1–2 pmol/30 µl/30-min sample) after 2 and 8 days of treatment was not significantly different from that in control animals, but the 21-day treatment gave significant rises in the extracellular GABA concentration (up to ∼6–8 pmol/30 µl/30-min sample). Both inhibitors gave similar results. Depolarisation with 100 m M K⁺ gave large increases in GABA release in control (∼20–60 pmol/30 µl/30-min sample) and treated animals. The 8- and 21-day-treated animals showed significant increases in the stimulated release compared with control animals (∼80–100 pmol/30 µl/30-min sample). Excluding Ca²⁺ had no significant effect on either basal or stimulated release. The significant increases in K⁺-evoked release of GABA show that the increased intracellular pool of GABA is available for release, and this may be related to the anticonvulsant action of these compounds.     

Chemical deacylation reduces the adhesive properties of proteolipid protein and leads to decompaction of the myelin sheath

Myelin proteolipid protein (PLP) contains thioester-bound, long-chain fatty acids which are known to influence the structure of the molecule. To gain further insights into the role of this post-translational modification, we studied the effect that chemical deacylation of PLP had on the morphology of myelin and on the protein's ability to mediate the clustering of lipid vesicles. Incubation of rat optic nerves in isoosmotic solutions containing 100 mM hydroxylamine (HA) pH 7.4 led to deacylation of PLP and decompaction of myelin lamellae at the level of the intraperiod line. Incubation of nerves with milder nucleophilic agents (Tris and methylamine) or diluted HA, conditions that do not remove protein-bound fatty acids, caused no alterations in myelin structure. Other possible effects of HA which could have affected myelin compaction indirectly were ruled out. Incubation of optic nerves with 50 mM dithioerythritol (DTE) also led to the splitting of the myelin intraperiod line and this change again coincided with the removal of fatty acids. In addition, the apparently compacted CNS myelin in the PLP-less myelin-deficient rat, like that in tissue containing deacylated PLP, was readily decompacted upon incubation in isoosmotic buffers, suggesting that the function of PLP as a stabilizer of the interlamellar attachment is, at least in part, mediated by fatty acylation. Furthermore, in contrast to the native protein, PLP deacylated with either HA or DTE failed to induce the clustering of phosphatidylcholine/cholesterol vesicles in vitro. This phenomenon is not due to side-effects of the deacylation procedure since, upon partial repalmitoylation, the protein recovered most of its original vesicle-clustering activity. Collectively, these findings suggest that palmitoylation, by influencing the adhesive properties of PLP, is important for stabilizing the multilamellar structure of myelin.