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Results for “Myelin Basic Protein”

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PubMed1986

Effect of chemical modifications of myelin basic protein on its interaction with lipid interfaces and cell fusion ability.

The ability of native and chemically modified myelin basic protein to induce fusion of chicken erythrocytes and to interact with lipids in monolayers at the air-water interface and liposomes was studied. Chemical modifications of myelin basic protein were performed by acetylation and succinylation: the positive charges of the native protein were blocked to an extent of about 90-95%. Cellular aggregation and fusion of erythrocytes into multinucleated cells was induced by the native myelin basic protein. This effect was diminished for both acetylated and succinylated myelin basic protein. Native myelin basic protein penetrated appreciably in sulphatide-containing lipid monolayers while lower penetration occurred in monolayers of neutral lipids. Contrary to this, both chemically modified myelin basic proteins did not show any selectivity to penetrate into interfaces of neutral or negatively charged lipids. The intrinsic fluorescence of the native and chemically modified myelin basic proteins upon interacting with liposomes constituted by dipalmitoylphosphatidycholine, glycosphingolipids, egg phosphatidic acid or dipalmitoylphosphatidyl glycerol was studied. The interaction with liposomes of anionic lipids is accompanied by a blue shift of the maximum of the native protein emission fluorescence spectrum from 346 nm to 335 nm; no shift was observed with liposomes containing neutral lipids. The acetylated and succinylated myelin basic proteins did not show changes of their emission spectra upon interacting with any of the lipids studied. The results obtained in monolayers and the fluorescence shifts indicate a lack of correlation between the ability of the modified proteins to penetrate lipid interfaces and the microenvironment sensed by the tryptophan-containing domain.

Acetylation↗

PubMed1989

Restoration of myelin formation by a single type of myelin basic protein in transgenic shiverer mice.

A minigene containing mouse cDNA coding for the smallest type of myelin basic protein and including the native promoter was constructed and used to produce transgenic shiverer mice. The hypomyelinating mouse, the shiverer, has a deletion in its myelin basic protein gene, lacks all four types of myelin basic protein in its myelin, and shows abnormal behavior such as violent tremors. Five of twenty-one transgenic shiverer mice showed recovered protein synthesis, compact myelin formation, and normal behavior. These results suggest that a single type of myelin basic protein restores myelin formation and returns the shivering phenotype to normal in the transgenic shiverer mouse.

Animals↗

PubMed1990

Distribution of myelin basic protein and P2 mRNAs in rabbit spinal cord oligodendrocytes.

Myelin basic protein (MBP) and P2 protein are small positively charged proteins found in oligodendrocytes of rabbit spinal cord. Both proteins become incorporated into compact myelin. We have begun investigations into the mechanisms by which MBP and P2 become incorporated into the myelin membrane. We find that P2, like the MBPs, is synthesized on free polysomes in rabbit spinal cord. Cell fractionation experiments reveal that rabbit MBP mRNAs are preferentially segregated to the peripheral myelinating regions whereas P2 mRNAs are predominantly localized within the perikaryon of the cell. In vitro synthesized rabbit MBP readily associates with membranes added to translation mixtures, whereas P2 protein does not. It is possible that P2 requires a "receptor" molecule, perhaps a membrane-anchored protein, for association with the cytoplasmic face of the myelin membrane.

Animals↗

PubMed1978

Immunocytochemical localization studies of myelin basic protein.

The location of myelin encephalitogenic or basic protein (BP) in peripheral nervous system (PNS) and central nervous system (CNS) was investigated by immunofluorescence and horseradish peroxidase (HRP) immunocytochemistry. BP or cross-reacting material could be clearly localized to myelin by immunofluorescence and light microscope HRP immunocytochemistry. Fine structural studies proved to be much more difficult, especially in the CNS, due to problems in tissue fixation and penetration of reagents. Sequential fixation in aldehyde followed by ethanol or methanol provided the best conditions for ultrastructural indirect immunocytochemical studies. In PNS tissue, anti-BP was localized exclusively to the intraperiod line of myelin. Because of limitations in technique, the localization of BP in CNS myelin could not be unequivocally determined. In both PNS and CNS tissue, no anti-BP binding to nonmyelin cellular or membranous elements was detected.

Animals↗

PubMed1999

A simple and rapid method for isolating myelin basic protein.

The conduction of impulses along axons of nerves is facilitated by the myelin sheath, composed of proteins and lipid. Myelin basic proteins (MBPs) are extrinsic membrane proteins that play an important role in the structural organization of the myelin sheath. In the central nervous system, MBPs account for 30-40% of total protein. The traditional method of MBP isolation involves the use of chloroform-ethanol, which would destroy the native form of MBP. A modified method for maintaining its native form was developed. The white matter of porcine brain was directly extracted by buffers containing different concentrations of sodium chloride owing to MBP solubilized at high concentration of NaCl. The MBP was further purified by cation exchange chromatography and buffers containing glycine and salts. Purified MBP were consistently obtained by this method.

Animals↗

PubMed2001

A distal upstream enhancer from the myelin basic protein gene regulates expression in myelin-forming schwann cells.

In peripheral nerves, large caliber axons are ensheathed by myelin-elaborating Schwann cells. Multiple lines of evidence demonstrate that expression of the genes encoding myelin structural proteins occurs in Schwann cells in response to axonal instructions. To gain further insight into the mechanisms controlling myelin gene expression, we used reporter constructs in transgenic mice to search for the DNA elements that regulate the myelin basic protein (MBP) gene. Through this in vivo investigation, we provide evidence for the participation of multiple, widely distributed, positive and negative elements in the overall control of MBP expression. Notably, all constructs bearing a 0.6 kb far-upstream sequence, designated Schwann cell enhancer 1 (SCE1), expressed at high levels in myelin-forming Schwann cells. In addition, robust targeting activity conferred by SCE1 was shown to be independent of other MBP 5' flanking sequence. These observations suggest that SCE1 will make available a powerful tool to drive transgene expression in myelinating Schwann cells and that a focused analysis of the SCE1 sequence will lead to the identification of transcription factor binding sites that positively regulate MBP expression.

Animals↗

PubMed1984

Mutual stimulation by phosphatidylinositol-4-phosphate and myelin basic protein of their phosphorylation by the kinases solubilized from rat brain myelin.

Myelin basic protein and phosphatidylinositol-4-phosphate are phosphorylated in vitro by ATP and solubilized rat brain myelin. When both substrates are present together, the rate of phosphorylation of each is increased about eight-fold. It appears likely that the phosphate turnover of myelin basic protein and of phosphatidylinositol-4-phosphate are coupled in vivo.

Animals↗

PubMed1993

Human T cell autoimmunity against myelin basic protein: CD4+ cells recognizing epitopes of the T cell receptor beta chain from a myelin basic protein-specific T cell clone.

We have investigated whether the normal immune system contains T cells that are able to recognize T cell receptor (TcR) determinants of autologous autoantigen-specific T cells. The T cell clone HW.BP3, specific for myelin basic protein (MBP) was isolated from a healthy donor. HW.BP3 is restricted by HLA-DR2a, and reacts to human MBP 139-153. The expressed alpha beta TcR genes of HW.BP3 were cloned and sequenced, and the sequences analyzed for potential T cell epitopes. Two synthetic peptides, one from the VDJ beta junctional (beta 1) and one from the V beta region (beta 2) of the TcR of HW.BP3, were used to select four TcR peptide-specific T cell lines from the donor of HW.BP3. All anti-TcR lines had the phenotype CD3+/CD4+/HLA-DR+/CD25+/CD45RO+, and recognized the antigen in the context of HLA-DR. Three anti-TcR lines, which had been selected for reactivity to peptide beta 1, recognized exclusively this peptide restricted by HLA-DR2b. One anti-TcR line, selected for peptide beta 2, responded to both peptides beta 1 and beta 2 when presented by autologous blood mononuclear cells, but not by HLA-DR2a- or HLA-DR2b-transfected L cells. All TcR peptide-specific T cell lines were efficiently cytotoxic. They specifically lysed autologous macrophages or HW.BP3 line cells in the presence of exogenous peptide antigen. In contrast, HW.BP3 did not present endogenous TcR peptides to the anti-TcR lines. The results demonstrate that the normal human immune system contains not only autoantigen-specific T cells, but also T cells that recognize antigenic determinants of autologous autoreactive TcR.

Amino Acid Sequence↗

PubMed1991

Regulation of myelin basic protein gene transcription in normal and shiverer mutant mice.

We describe the measurement of myelin basic protein gene transcription rate, and of the accumulation of both mature mRNA and total transcripts from the myelin basic protein gene in brains from mice of wild-type and homozygous shiverer genotypes at several ages spanning postnatal development. In wild-type brains the accumulation of total transcripts as well as mature mRNA, and the transcription rate, all follow the same general pattern of rising sharply from a low level at birth to a peak at 20 days, and continuing at a somewhat reduced level into adulthood. Thus a major factor in the developmental regulation of myelin basic protein expression is the control of transcription rate. The shiverer mutation consists of a deletion of the 3' end of the myelin basic protein gene which completely prevents production of mature mRNA and protein, and results in severe dysmyelination and a trembling behavior. In shiverer brains, the transcription rates for the intact 5' end of the gene follow closely those seen in wild-type animals up to the age at which maximal myelination normally occurs. Total myelin basic protein transcripts follow a similar profile but at less than 5% the level seen in wild-type, and, as expected, no mature mRNA is detected. Thus the shiverer deletion does not remove information required for efficient, developmentally regulated transcription, and the low level of myelin basic protein gene transcripts in this mutant must be a result of their reduced stability. A higher than normal myelin basic protein gene transcription rate in older shiverer animals raises interesting questions regarding the regulatory mechanisms controlling myelinogenesis.

Animals↗

PubMed1984

Surface accessibility of 13C-labeled lysine residues in membrane-bound myelin basic protein.

Surface-exposed regions of membrane-bound myelin basic protein--the major extrinsic membrane protein of central nervous system myelin--have been implicated as possible antigenic sites in diseased myelin. With the goal of determining the extent and nature of these regions, we have prepared basic protein modified with 13CH3-enriched acetyl groups at 7 of its 13 lysine residues. The resulting protein was placed in a membrane environment and studied by NMR spectroscopy to determine the location and rates of molecular motion of the labeled side chains with respect to lipid bilayers of the membrane. When 13C NMR spectra were obtained of the acetylated protein bound to multilamellar vesicles prepared from dimyristoylphosphatidic acid in the gel state (T = 33 degrees C), conditions under which reduced motion in the lipid bilayer broadens methylene and methyl 13C resonances of the membrane beyond detection (i.e. greater than 75-100 Hz), line widths of membrane-bound protein were measured to be 7.8 Hz, an increase of 4 Hz versus free protein. A reduction of 25-30% in integrated intensity observed in protein acetyl resonances upon membrane interaction was shown to be attributable to a population of protein-aggregated liposomes whose resonances were similarly too broad to be observed. Thus, the epsilon-acetyllysyl probes distributed throughout the protein do not penetrate the dimyristoylphosphatidic acid bilayer, but must reside in the interstitial aqueous spaces at or between membrane surfaces. These findings suggest an overall surface accessibility of membrane-bound myelin basic protein and are therefore incompatible with a model for the protein involving membrane-embedded loops or regions of functional significance.

Acetic Anhydrides↗

PubMed1998

The expression of myelin basic protein exon 1 and exon 2 containing transcripts during myelination of the neonatal rat spinal cord--an in situ hybridization study.

During myelination, myelin proteins are expressed in a highly coordinated sequence. Of the four major isoforms of myelin basic protein, the proportion of the 21.5kDa and 17 kDa isoforms (which contain sequences encoded by myelin basic protein [MBP] exon 2) is enriched during active myelination in the mouse, suggesting that the alternative splicing of MBP transcripts containing exon 2 information is developmentally regulated. In this study, we compare the expression of MBP exon 1 and MBP exon 2 mRNAs in the neonatal rat spinal cord to establish whether developmental regulation of exon 2 mRNAs occurs in the rat in a manner similar to that previously described in the mouse. The expression of MBP mRNAs, together with that of proteolipid protein (PLP/DM-20) mRNA, in the developing white matter tracts increased dramatically between P7 and P10, corresponding to an increase in the extent of myelination. High levels of expression of each mRNA species examined were maintained between P10 and P14 as myelination proceeded. At P21, the expression of MBP exons 1 and 2 was reduced in the ventrolateral funiculi but was maintained at high levels in the dorsal funiculus. At P45, a further downregulation of both MBP mRNAs was apparent. By contrast, high levels of PLP/DM-20 expression were maintained from P10 onwards. In the grey matter, expression of MBP and PLP/DM-20 mRNAs increased more gradually and peak expression occurred later than in the white matter tracts. In this study, we therefore provide a description of myelin protein gene expression during post-natal development of the rat spinal cord. We have also shown that in the rat spinal cord, changes in the levels of MBP exon 2 expression associated with myelination reflect the changes of all MBP transcripts.

Alternative Splicing↗

PubMed1982

The polyadenylated RNA directing the synthesis of the rat myelin basic proteins is present in both free and membrane-bound forebrain polyribosomes.

Free and membrane-bound polyribosomes were isolated from the forebrain of actively myelinating 24-day-old rats. The poly(A)+ RNA (polyadenylated RNA) extracted from both fractions was translated in vitro in reticulocyte lysates [Hall & Lim (1981) Biochem. J. 196. 327-336] in the presence or absence of a heterologous microsomal membrane fraction from dog pancreas. The rat myelin basic proteins synthesized in vitro were isolated by CM-cellulose chromatography and by immunoprecipitation with purified anti-(myelin basic protein) antibody. The large (mol.wt. 18 500) and small (mol.wt. 16 000) myelin basic proteins were translational products of poly(A)+ RNA from both free and membrane-bound polyribosomes. The identity of the myelin basic proteins was verified by analysis of peptides generated by the cathepsin D digestion of the immunoprecipitated proteins synthesized in vitro, in comparison with authentic rat myelin basic proteins. Although several other translational products of membrane-bound polyribosomal poly(A)+ RNA were modified when microsomal membranes were present during translation, molecular weights of the myelin basic proteins themselves were unchanged. The myelin basic proteins synthesized in vitro also did not differ significantly in size from the authentic myelin basic proteins, indicating that these membrane proteins are unlikely to be synthesized as substantially larger precursor molecules. The presence of the specific mRNA species on both free and membrane-bound polyribosomes is compatible with the extrinsic location of the myelin basic proteins on the cytoplasmic surface of the myelin membrane.

Animals↗

PubMed1991

Identification of the new isoforms of mouse myelin basic protein: the existence of exon 5a.

Myelin basic protein (MBP) is a major constituent in the myelin of the CNS. In mice, five forms of MBPs (14 kDa, two types of 17 kDa, 18.5 kDa, and 21.5 kDa) encoded by separate mRNAs have been identified based on cDNA cloning studies. These mRNAs are considered to be produced by alternative splicing from a single gene composed of seven exons. Here we report the existence of two novel MBP mRNAs encoding 19.7-kDa and 21-kDa MBPs identified by cDNA cloning using the polymerase chain reaction. Both of these MBPs contain a sequence of a previously unidentified exon of 66 nucleotides, which was mapped to be just 5' of exon 5 in the MBP gene. MBP mRNAs containing this novel exon (exon 5a) belong to a minor population in the whole brain and PNS and are somewhat enriched in the spinal cord. Exon 5a encodes a very hydrophobic segment rich in valine residues, which presumably forms a beta-pleated sheet.

Animals↗

PubMed1988

Expression and localization of myelin basic protein in oligodendrocytes and transfected fibroblasts.

Myelin basic protein (MBP) is a major structural component of myelin. It is expressed exclusively in myelinating glia (oligodendrocytes in the CNS and Schwann cells in the PNS) and is localized to the cytoplasmic surface of the plasma membrane and myelin membrane produced by these cells. The work described here concerns the mechanism of plasma membrane localization of MBP in myelinating glial cells and whether it involves differentiated functions specific to these cells or general functions of plasma membrane assembly common to all cells. To this end, the subcellular localization of endogenous MBP in mouse oligodendrocytes was compared with that of transiently expressed MBP in monkey fibroblasts (Cos-1 cells) transfected with an MBP expression vector containing cDNA for rat 14K MBP. The steady-state levels of MBP-specific RNA and of MBP polypeptide expressed in the transfected fibroblasts were comparable to the levels expressed in oligodendrocytes in primary culture. MBP localization was analyzed in whole cells by immunofluorescence and in specific intracellular compartments by subcellular fractionation. The results show that MBP expressed in wild-type oligodendrocytes is localized to the plasma membrane. In contrast, MBP expressed in transfected fibroblasts appears dispersed in the cytoplasm and is distributed uniformly among the various subcellular fractions.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1979

Radioimmunoassay of serum myelin basic protein and its application to patients with cerebrovascular accident.

Myelin basic protein-like immunoactivity was measured in the serum of patients after cerebrovascular accident (CVA) using a double antibody radioimmunoassay for myelin basic protein with a detection limit of 3 ng/ml serum. For up to 6 days after ictus, serum myelin basic protein levels in patients with severe CVA and patients who died as a result of CVA were significantly greater than those in control patients, patients with moderate CVA and patients surviving CVA. All patients with serum myelin basic protein levels greater than the range found in control subjects subsequently died. Serial dilutions of positive sera suggested that the immunoactivity differs from authentic myelin basic protein and may represent breakdown products of the protein. Serum from some patients with a previous history of moderate CVA had myelin basic protein binding activity consistent with the presence of antibodies to the protein.

Cerebrovascular Disorders↗

PubMed1994

Immunohistochemical detection of myelin basic protein is a sensitive marker of myelination in second trimester human fetal spinal cord.

The Luxol fast blue (LFB) technique is widely used for the assessment of myelination. Lectin histochemistry using peanut agglutinin (PNA) has also been employed for this purpose. Recently, immunohistochemical methods using antibodies to several myelin-related proteins have been adopted to study myelination in humans. However, the relative sensitivities of these different methods for the detection of early myelination in the human fetal central nervous system have not been determined. Vibratome sections of cervical spinal cord from 15 human abortuses ranging in age from 15 to 24 gestational weeks (GW) were probed with immunohistochemical methods using antibodies to myelin basic protein (MBP), 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase), and myelin-associated glycoprotein (MAG). In addition, LFB and PNA histochemistry was employed. The degree of myelination observed in immunohistochemically stained sections was compared to that found in corresponding LFB- and PNA-stained paraffin-embedded tissues. The intensity of myelination was graded by two observers on a scale of 0 (none), +1 (mild), +2 (moderate), and +3 (marked). At all ages examined, the MBP immunohistochemical method revealed more myelin than LFB or MAG staining. CNPase could not be reliably detected until after 18 GW. Peanut agglutinin stained myelin, but subpial astrocytes and the intervening neuropil were also stained. These results suggest that MBP is a more sensitive marker for early human fetal myelination than CNPase, MAG, PNA or LFB.

Abortion, Induced↗

PubMed1975

Experimental autoimmune encephalomyelitis in mice: immunologic response to mouse spinal cord and myelin basic proteins.

It was confirmed that experimental autoimmune encephalomyelitis EAE, could be induced in SJL/J mice with mouse spinal cord homogenate. It was shown that induction of EAE in mice was critically dependent on the concentration of pertussis vaccine. The encephalitogen present in mouse brain was the basic protein of myelin. The smaller form of the mouse and rat basic proteins induced EAE; thus the mouse like the rat responds to determinants other than the "tryptophan region," which induced EAE in guinea-pigs. Mice with EAE developed a cell-mediated immune response to myelin basic protein, as judged by inhibition of peritoneal cell migration. However, levels of antibody to mouse basic protein were low, as judged by radioimmunoassay. The establishment of this autoimmune disease model in the mouse will allow the application of well established techniques for the analysis of the immunologic mechanisms leading to disease manifestation.

Animals↗

PubMed1995

A 39-kD DNA-binding protein from mouse brain stimulates transcription of myelin basic protein gene in oligodendrocytic cells.

The MB1 regulatory sequence of the myelin basic protein (MBP) gene spanning between nucleotides -14 to -50 with respect to the transcription start site is critical for cell type-specific transcription of the MBP gene, which encodes the major protein component of myelin sheath in cells derived from the central nervous system (CNS). This regulatory sequence has the ability to interact with a developmentally controlled DNA-binding protein from mouse brain that stimulates transcription of MBP promoter in an in vitro system (Haas, S., J. Gordon, and K. Khalili. 1993. Mol. Cell. Biol. 13:3103-3112). Here, we report the purification of a 39-kD protein from mouse brain tissue at the peak of myelination and MBP production that binds to the MB1 regulatory motif. Following partial amino acid sequence analysis, we have identified a complementary DNA encoding a 39-kD DNA-binding protein called pur alpha. Expression of pur alpha cDNA in the prokaryotic and eukaryotic cells resulted in the synthesis of a protein with characteristics similar to the purified brain-derived 39-kD protein in band shift competition assays. Cotransfection of the recombinant pur alpha expressor plasmid with MBP promoter construct indicated that Pur alpha stimulates transcription of the MBP promoter in oligodendrocytic cells, and that the nucleotide sequence required for binding of the 39-kD Pur alpha to DNA within the MB1 region is crucial for this activity. Moreover, transient expression of Pur alpha caused elevation in the level of endogenous MBP RNA in oligodendrocytic cells. Thus, Pur alpha, a sequence-specific DNA-binding protein upon binding to MB1 regulatory region may play a significant role in determining the cell type-specific expression of MBP in brain.

Animals↗