Selective extraction, solubilization, and reversed‐phase high‐performance liquid chromatography separation of the main proteins from myelin using tetrahydrofuran/water mixtures

Díaz, R. S.; Regueiro, Pilar; Monreal, J.; Tandler, C. J.

doi:10.1002/jnr.490290113

Cited by 11 publications

(5 citation statements)

References 16 publications

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“…By contrast with a recent study that used purified and recombinant proteins as target Ags (30), the absence of reactivity against myelin proteins observed in our study was probably due to the weak representation of these proteins in whole brain tissue samples. In addition, SDS extraction allowed the solubilization of MBP and a large proportion of MOG, but without organic extraction, proteolipid lipoprotein is faintly represented (51)(52)(53). Nevertheless, our method suggests that various Ags support specific immune recognition in MS.…”

Section: Discussionmentioning

confidence: 99%

Distortion of the Self-Reactive IgG Antibody Repertoire in Multiple Sclerosis as a New Diagnostic Tool

Lefranc¹,

Almeras²,

Dubucquoi³

et al. 2004

The Journal of Immunology

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To date, none of the myelin-associated Ag targets definitively discriminates between the immune response observed in multiple sclerosis (MS) patients and healthy subjects. However, it has been shown recently that analysis of global immune Ab profiles such as natural autoantibody reactivities can help to distinguish between normal individuals and patients suffering from various immune diseases. The aim of our study was to compare the global IgG immune response against brain self-Ags in sera from 82 MS patients and 27 healthy subjects. The analysis of the immune profiles was performed by Western blotting, and data were subjected to linear discriminant analysis. Particular patterns of IgG reactivity were found in healthy subjects, Sjögren patients, and MS patients. Moreover, this approach separated the three clinical forms of MS with a high concordance rate with the clinical data (κ value, 77.8%). Our study suggests, for the first time, that serum IgG Ab repertoires are able to distinguish MS patients. In addition, our data suggest that patterns of IgG reactivity could model the pathological processes underlying the various forms of MS. Further characterization of such discriminant Ags could provide useful information regarding their potent role in pathogenesis or regulatory processes in MS.

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Section: Discussionmentioning

confidence: 99%

Distortion of the Self-Reactive IgG Antibody Repertoire in Multiple Sclerosis as a New Diagnostic Tool

Lefranc¹,

Almeras²,

Dubucquoi³

et al. 2004

The Journal of Immunology

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“…1). The reason is because the mixture of THF with water in several proportions extracts proteins from white matter in a quantitative and selective manner (Diaz et al, 1991).…”

Section: Discussionmentioning

confidence: 99%

“…Basic protein (BP, Mr 18,500) was extracted from bovine brain white matter according to Oshiro and Eylar (1970) and purified to homogeneity by reversed-phase high-performance liquid chromatography using the conditions defined for myelin proteins by Diaz et al (1991). Small unilamellar vesicles (SUVs) were prepared from TLE or DML at a concentration of 5 mg lipid/ml in buffer 100 mM NaCl, 10 mM HEPES, pH 7.4 by the conditions shown elsewhere (Wood and Moscarello, 1989).…”

Section: Aggregation Of Liposomes With Basic Proteinmentioning

confidence: 99%

Preparation of a protein‐free total brain white matter lipid fraction: Characterization of liposomes

Díaz¹,

Monreal²,

Regueiro³

et al. 1992

J of Neuroscience Research

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A method of preparing a total lipid extract (TLE), free of protein, by extracting brain white matter with tetrahydrofuran is presented. The optimal conditions of extraction were found to be 50 ml of THF per gram of lyophilized tissue, though fresh tissue can also be used if larger volumes of solvent are employed. The method allowed, in a short time and in a single step, a yield of TLE of 50% on a dry weight basis. Its analytical characterization revealed a qualitative and quantitative composition very similar to the lipid composition of CNS myelin, including all the phospholipid and galactolipid species, cholesterol and gangliosides, but it contained only traces (0.1%) of protein. TLE has been used to prepare liposomes, either multilamellar (MLVs) or unilamellar (LUVs, SUVs), characterized by freeze ‐fracture electron microscopy. A multilayered, heterogeneous population of liposomes is observed in the MLVs preparation. When these samples were submitted to a freezing and thawing procedure the resulting liposomes were single‐walled, and their intravesicular volume was increased. They were quite impermeable to the mono‐valent cation 86Rb+ and, by contrast rather permeable to 45Ca+ +. Their complex lipid composition, together with their permeability properties and their response to ionophores, make them very useful to study protein‐lipid interactions occurring within the myelin membrane as well as the functional properties of myelin proteins in reconstitution experiments.

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“…To confirm the incorporation of protein into giant proteoliposomes, they were prepared with the water-soluble form of proteolipids, as described later. A homogeneous preparation of myelin basic protein (MBP) was obtained according to the technique of DIaz et al (1991) and was reconstituted in liposomes of TLE according to the method of GarcIa-Segura et al (1986).…”

Section: Myelin Purification and Preparation Of Vesiclesmentioning

confidence: 99%

Preparation of Giant Myelin Vesicles and Proteoliposomes to Register Ionic Channels

Regueiro

Monreal

Díaz

et al. 1996

Journal of Neurochemistry

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Myelin vesicles, reconstituted liposomes with proteolipid protein (PLP), the main protein component of myelin, and electrophysiological patch‐clamp are potentially powerful tools to study the role of myelin in functional ionic channels. However, technical difficulties in the vesiculation of myelin and the small size of the vesicles obtained do not permit the application of micropipettes for current recordings. From a suspension of purified myelin we have prepared oligolamellar vesicles (mean diameter of 144 nm) using the so‐called French pressure system. From this preparation we obtained giant myelin vesicles ∼10 µm in mean diameter, using a dehydration‐rehydration procedure. Qualitative analysis of proteins by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis revealed no significant loss of any component in these vesicles due to pressure, in comparison with non‐vesiculated myelin. A way of preparing giant liposomes of ∼80–100 µm and proteoliposomes of ∼30 µm in mean diameter, using the same dehydration‐rehydration procedure, is also reported. Reconstitution of purified PLP in giant liposomes was confirmed by fluorescent labeling of PLP and by fluorescence microscopy. The current recordings from these vesicles prove the validity of these methods and provide significant evidence of the existence of ionic channels in myelin membranes and the possibility that PLP functions as a channel. The physiological significance and characterization of these channels remain yet unresolved. These results have a special significance for elucidating the molecular role of myelin in the regulation of neural activity and in the brain ion microenvironment.

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Selective extraction, solubilization, and reversed‐phase high‐performance liquid chromatography separation of the main proteins from myelin using tetrahydrofuran/water mixtures

Cited by 11 publications

References 16 publications

Distortion of the Self-Reactive IgG Antibody Repertoire in Multiple Sclerosis as a New Diagnostic Tool

Distortion of the Self-Reactive IgG Antibody Repertoire in Multiple Sclerosis as a New Diagnostic Tool

Preparation of a protein‐free total brain white matter lipid fraction: Characterization of liposomes

Preparation of Giant Myelin Vesicles and Proteoliposomes to Register Ionic Channels

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