Glycolipids and myelin proteins in human oligodendrogliomas

We studied myelin proteins and glycolipids in 24 human oligodendrogliomas (16 pure, eight mixed), including two grade I, 13 grade II, five grade III, and four grade IV Tumours with a 1b ganglioside content (GD1b, GT1b and GQ1b) over 30% of total gangliosides occur more frequently in the WHO grade I II and (47%) and grade III (40%) than in the grade IV (25%) group; there was no difference in the amounts of total ganglioside or individual gangliosides between pure and mixed oligodendrogliomas. The presence of 6-LM1 correlated with higher grades of tumours (² p0.02); however, 3-LM1 and total neolacto-series gangliosides did not correlate with grade. Immunohistochemical studies of oligodendrocyte and myelin markers (GalCer, sulfatide, 2,3-cyclic nucleotide phosphodiesterase, myelin basic protein and proteolipid protein) using specific antibodies showed only a very small proportion of tumour cells staining. These data do not support the hypothesis that tumours classified as oligodendrogliomas are derived from mature oligodendrocytes.     

Increased susceptibility to degradation by trypsin and subtilisin of in vitro peroxidized myelin proteins

We examined the possibility that the peroxidative damage to central nervous system myelin produced by reactive oxygen species (ROS), could modify the susceptibility of its proteins to the proteolytic action of proteases such as trypsin and subtilisin. Purified myelin membranes obtained from adult rat brains were in vitro peroxidized by two non-enzymatic systems: Fe³⁺ plus ascorbic acid and Cu²⁺ plus hydrogen peroxide. Myelin proteins were severely affected by peroxidation. There was an increase in the amount of carbonyl groups (CO), accompanied by and enhanced susceptibility to degradation by trypsin and subtilisin of myelin basic proteins (MBP) and of the major proteolipid protein (PLP). The effect upon the degradation of myelin protein is a possible consequence of the appearance in the structure of myelin proteins of peroxidative modifications that contribute to the recognition by proteolytic enzymes. This hypothesis is supported by the fact that if peroxidation of myelin membranes is done in the presence of EDTA, both CO formation and increased sensitivity to enzymatic breakdown disappear. These results suggest that the appearance of abnormal post-translational modifications in the myelin membrane produced by peroxidation could constitute a putative mechanism of modulating the capacity of myelin proteins to be metabolized by proteases.     

Phosphorylation of myelin proteins: Recent advances

Since it was first described 25 years ago, phosphorylation has come to be recognized as a widespread and dynamic post-translational modification of myelin protein. In this review, the phosphorylation characteristics of myelin basic protein, protein zero (P₀), myelin-associated glycoprotein and 2′3′ cyclic nucleotide 3′-phosphodiesterase are summarized. Emphasis is placed on recent advances in our knowledge concerning the protein kinases involved and the sites, of phosphorylation in the amino acid sequences, where known. The possible roles of myelin protein phosphorylation in modulating myelin structure, the process of myelin assembly and mediation of signal transduction events are discussed. Special issue dedicated to Dr. Marion E. Smith.     

A 42 K protein of chick sciatic nerve is immunologically related to PO protein of peripheral nerve myelin

The major protein (PO) in PNS myelin is an integral membrane glycoprotein with a molecular weight of about 30 K. The level of PO protein in the developing sciatic nerve of the chicken was monitored by a solid-phase immunoassay and densitometry of Coomassie blue stained polyacrylamide gels. The most rapid rate of accumulation of PO protein occurred after 16 days of embryonic development. In addition to the 30 K PO protein, a number of higher molecular weight proteins could be distinctly detected by immunoblotting. Amongst these high molecular weight proteins, a species with an apparent molecular weight of 42 K was specifically immunostained with epitope-selected polyclonal antibodies against PO protein. This 42 K protein could be first detected after 16 days of embryonic development and increased rapidly following the pattern of myelination in the sciatic nerve. The enzyme endoglycosidase F, which specifically removes N-asparagine linked high mannose and complex carbohydrates from glycoproteins, converted the PO and 42 K proteins to lower molecular weight forms, which could be specifically immunostained by epitope selected polyclonal antibodies to the PO protein. Subcellular fractionation of the 17-day embryonic nerve demonstrated that the 42 K protein was enriched in myelin and microsomal subfractions relative to the total homogenate. These results indicate that the 42 K immuno-crossreactive protein might be chemically and functionally related to the PO protein of the PNS myelin.Abbreviations used CNP 2–3-cyclic-nucleotide 3-phosphodiesterase (EC 3.1.4.37) - EDTA ethylene(dinitrilo)tetraacetic acid - Endo F endo--N-acetylglucosaminidase F (EC 3.2.1.96) - NADPH Cyt. Red. NADPH-cytochrome C reductase (EC 1.6.2.4) - 5-NPase 5-ribonucleotide phosphohydrolase (EC 3.1.3.5) - PNS peripheral nervous system - SDS sodium dodecyl sulfate - TPPase thiamine pyrophosphatase (EC 3.1.6.X)     

Basic proteins of rodent peripheral nerve myelin: immunochemical identification of the 21.5K, 18.5K, 17K, 14K, and P2 proteins

Abstract: Proteins in peripheral nervous system and central nervous system myelin and homogenates of sciatic nerve and brain from young and adult mice and rats were characterized with affinity-purified anti-P₂ and anti-myelin basic protein sera after electrophoretic transfer from sodium dodecyl sulfate-polyacrylamide gels to nitrocellulose sheets. Using this method we have identified a component of rodent peripheral nervous system myelin as P₂ protein. Peripheral nervous system myelin also showed the presence of four basic proteins in addition to P₂ protein. These were found to be analogous to the 14, 17, 18.5, and 21.5K species found in the central nervous system myelin. A number of high-molecular-weight proteins were also detected with anti-myelin basic protein serum in peripheral nervous system, as well as central nervous system myelin. In addition, we report the presence of a high-molecular-weight P₂ cross-reactive protein in rodent brain stem homogenates, but not in central nervous system myelin.     

Specificity of zinc binding to myelin basic protein

Z²⁺ appears to stabilize the myelin sheath but the mechanism of this effect is unknown. In a previous report we have shown that zinc binds to CNS myelin basic protein (MBP) in the presence of phosphate and this results in MBP aggregation. For this paper we used a solid phase zinc blotting assay to identify which myelin proteins bind zinc. MBP and a 58 kDa band were found to be the major targets of⁶⁵Zn binding. Moreover, using fluorescence, light scattering and electron microscopy we investigated the binding of zinc and other cations to purified MBP in solution. Among the cations tested for their ability to interfere with the binding of zinc, the most effective were cadmium, mercury and copper, but only cadmium and mercury increased the scattering intensity, whereas MBP aggregation was not inhibited by copper ions. Thus, the effect of zinc on the formation of MBP clusters seems to be specific.     

Triton X-100 extractions of central nervous system myelin indicate a possible role for the minor myelin proteins in the stability of lamellae

Isolated CNS myelin membranes were extracted with Triton X-100 under conditions previously established for the isolation of cytoskeletal proteins. Treated myelin retained much of its characteristic lamellar structure despite the removal of most of the major myelin basic protein (18.5 kDa) and the proteolipid protein, which together normally constitute 60% of the total myelin protein. The SDS-PAGE profile of this extract residue demonstrated an enrichment in proteins of Mr 30 to 60 kilodaltons (the Wolfgram group). The major myelin proteins were identified by antibodies on Western immunoblots, as were the 23-cyclic nucleotide 3-phosphodiesterase (CNP), actin, tubulin, myelin-associated glycoprotein (MGP) and the 21.5 kDa MBP. The overall behavior of CNP, the 21.5 kDa MBP, MGP and tubulin towards Triton extraction is reminiscent of the behavior of other membrane-skeletal complexes, supporting the idea that these and other minor myelin proteins might be part of heteromolecular complexes with interactions spanning several lamellae of the myelin sheath.     

Bimodal occurrence of aspartoacylase in myelin and cytosol of brain

The growing use of N-acetylaspartate as an indicator of neuronal viability has fostered interest in the biological function(s) of this unusual amino acid derivative. In considering the various physiological roles that have been proposed for this relatively abundant molecule one is obliged to take into account its unusual metabolic compartmentalization, according to which synthesis and storage occur in the neuron and hydrolytic cleavage in the oligodendrocyte. The latter reaction, catalyzed by aspartoacylase (ASPA), produces acetyl groups plus aspartate and has been proposed to occur in both soluble and membranous subfractions of white matter. Our study supports such bimodal occurrence and we now present immunoblot, proteomic, and biochemical evidence that the membrane-bound form of ASPA is intrinsic to purified myelin membranes. This was supported by a novel TLC-based method for the assay of ASPA. That observation, together with previous demonstrations of numerous lipid-synthesizing enzymes in myelin, suggests utilization of acetyl groups liberated by myelin-localized ASPA for lipid synthesis within the myelin sheath. Such synthesis might be selective and could explain the deficit of myelin lipids in animals lacking ASPA.     

Antigenic sites common to major fish myelin glycoproteins (IP) and to major tetrapod PNS myelin glycoprotein (Po) reside in the amino acid chains

The major myelin glycoproteins in the CNS and PNS of trout (IP) were enzymatically deglycosylated with endoglycosidase F (Endo F) and examined by electro-immunoblotting. Following carbohydrate removal and loss of concanavalin A affinity each of the four IP components underwent a similar reduction in molecular size, corresponding to approximately 3,000 daltons. Immunological cross-reactivities with anti-bovine P_o or anti-trout IP2 antibodies, were however fully retained by the Endo F cleavage products. This strongly implies that the antigenic sites shared by the mammalian P_o protein and the various intermediate glycoproteins of trout CNS and PNS are located in the protein portion. Immunoblot analysis of the PNS myelin proteins from various species of the major vertebrate classes with anti-trout IP2 antiserum revealed striking differences in the immunological properties of the individual P_o components which were not detected when anti-bovine P_o antiserum was used as a probe.     

The use of reaction timecourses to determine the level of minor contaminants in enzyme preparations

Enzyme mutagenesis is a commonly used tool to investigate the structure and activity of enzymes. However, even minute contamination of a weakly active mutant enzyme by a considerably more active wild-type enzyme can partially or completely obscure the activity of the mutant enzyme. In this work, we propose a theoretical approach using reaction timecourses and initial velocity measurements to determine the actual contamination level of an undesired wild-type enzyme. To test this method, we applied it to a batch of the Q215A/R235A double mutant of orotidine 5′-monophosphate decarboxylase (OMPDC) from Saccharomyces cerevisiae that was inadvertently contaminated by the more active wild-type OMPDC from Escherichia coli. The enzyme preparation showed significant deviations from the expected kinetic behavior at contamination levels as low as 0.093 mol%. We then confirmed the origin of the unexpected kinetic behavior by deliberately contaminating a sample of the mutant OMPDC from yeast that was known to be pure, with 0.015% wild-type OMPDC from E. coli and reproducing the same hybrid kinetic behavior.     

利用RAPD分析鉴定花椰菜杂种纯度

应用随机扩增多态性DNA(RAPD)分析方法,鉴定花椰菜F1代杂种纯度。筛选出20个10bp随机引物对杂种F1代和父母本基因组DNA进行RAPD分析,共获得扩增片段124条,分子量在0.3-3kb之间,其中2个引物S120和S174可用来鉴定杂种纯度。RAPD分析结果与田间形态鉴定结果基本一致,表明RAPD分析方法适用于花椰菜杂种的纯度鉴定。     

Vulnerability of myelin development of the chick to the herbicide 2,4-dichlorophenoxyacetic butyl ester

Fertilized hens' eggs were treated externally with 2,4-Dichlorophenoxyacetic butyl ester (2,4-D b.e.) (3.1 mg/egg) immediately before starting incubation, and after different times of incubation (5, 10 and 15 days). Controls were treated externally with ether. Hatchability studies demonstrated that fetotoxic effects of 2,4-D b. e. were similar on the 0, 5 and 10 incubation day, but the 15 Day Group improved the hatching percentage. One day after hatching, chicks were decapitated, and CNS tissue was dissected. Myelin markers, as cerebrosides and CNP, were determined in cerebrum, cerebellum, brain stem and spinal cord of the four groups. They were reduced in cerebrum and brain stem of the 0, 5 and 10 Day Groups, but in the 15 Day Group they were in normal levels. Cerebellum presented normal myelin marker contents in each group studied, while spinal cord only presented decreased marker contents in the 5 Day Group. UDP galactose-ceramide galactosyl transferase (EC 2.4.1.45) activity was reduced in whole brain of chicks born from eggs treated preincubation. The results show the importance of time drug application and suggest that the vulnerable period in CNS development includes proliferation and development of myelin forming cells. Among CNS regions, cerebrum and brain stem seem to be the most vulnerable to the toxic action of 2,4-D b.e. in the chick.     

Characterization of guanylyl cyclase in purified myelin

This study was undertaken to characterize the enzymatic properties of the particulate guanylyl cyclase previously shown to be present at a high level of activity in purified rat brain myelin. Significant activation was achieved by both Lubrol-PX and Triton X-100, the latters being somewhat more effective. A pH optimum of 7.8 was observed, compared to 7.4 for microsomes. Employing 1.2 mM GTP with 1% Triton X-100, linearity of response was observed up to 60 min and approximately 1.2 mg of myelin protein. Kinetic analysis revealed K_m values of 0.258 mM and 0.486mM for myelin and microsomes, respectively, similar values being obtained by Lineweaver-Burke analysis or Direct Linear Plot. V_max values were 20 and 266 pmol/mg protein/min for myelin and microsomes, respectively. Washing of the myelin with 0.5 M NaCl or 0.1% Na taurocholate did not remove a significant amount of guanylyl cyclase activity, indicating the enzyme to be intrinsic to the myelin sheath. Special issue dedicated to Dr. Marion E. Smith.     

Proteomic analysis of nuclear factors binding to an intronic enhancer in the myelin proteolipid protein gene

The myelin proteolipid protein gene ( Plp1 ) encodes the most abundant protein found in CNS myelin, accounting for nearly one-half of the total protein. Its expression in oligodendrocytes is developmentally regulated – peaking during the active myelination period of CNS development. Previously, we have identified a novel enhancer (designated ASE) in intron 1 DNA that appears to be important in mediating the surge of Plp1 gene activity during the active myelination period. Evidence suggests that the ASE participates in the formation of a specialized multi-protein/DNA complex called an enhanceosome. The current study describes an optimized, five-step, DNA affinity chromatography purification procedure to purify nuclear proteins from mouse brain that bind to the 85-bp ASE sequence, specifically. Electrophoretic mobility shift assay analysis demonstrated that specific DNA-binding activity was retained throughout the purification procedure, resulting in concomitant enrichment of nucleoprotein complexes. Identification of the purported regulatory factors was achieved through mass spectrometry analysis and included over 20 sequence-specific DNA-binding proteins. Supplementary western blot analyses to determine which of these sequence-specific factors are present in oligodendrocytes, and their developmental and regional expression in whole brain, suggest that Purα and Purβ rank highest among the candidate factors as constituents of the multi-protein complex formed on the ASE.     

Minireview: Autoimmune responses to myelin proteolipid protein

The authors present a brief historical sketch of the development of our understanding of immune responses to myelin proteolipid protein (PLP) and the acceptance of PLP as a potent antigen in the induction of experimental allergic encephalomyelitis (EAE). The distinct characteristics of the PLP molecule that may contribute to complex immune responses to this protein are reviewed and these responses are compared with those to MBP, both in the pathology of EAE and at the level of the T cell. Recent evidence demonstrating differences between T cell responses to PLP and MBP is reviewed. Finally, the potential contribution of immune responses to PLP in human diseases, particularly mutiple sclerosis (MS), that have been identified to date are then summarized.For the authors to write a review on PLP and its role in EAE without Marjorie is like their sailing a ship without a captain, compass or rudder. This review is largely based on work and ideas generated in Marjorie's laboratory, but it was prepared without her input. Consequently, it lacks her meticulous reflection on the structure of each of its sentences and on the use of each word. Papers written with Marjorie are usually honed to near perfection late into the evening at her kitchen table in Newton, where food, ideas, and warmth abound, and where her very patient and accommodating husband Sidney and a demanding but lovable canine are close at hand. Writing this essay gave the authors a chance to recognize our scientific forebears, to consider where we are at this point and to contemplate our future directions in studying immune responses to PLP. We are, indeed, very grateful and indebted to Marjorie for her generous personal and scientific support, wise guidance, inspiration, strength, energy and, most importantly, friendship. Marjorie, we thank you, you are our role model, and we affectionately anticipate many more years of continued collaboration with you.Abbreviations used in this paper CNS central nervous system - EAE experimental allergic encephalomyelitis - MBP myelin basic protein - MHC major histocompatibility complex - MOG myelin oligodendrocyte cyte glycoprotein - MS multiple sclerosis - PLP myelin proteolipid protein - PNS peripheral nervous system - TcR T cell receptor Special issue dedicated to Dr. Marjorie B. Lees.     

Binding of soluble myelin-associated glycoprotein to specific gangliosides induces the association of p75NTR to lipid rafts and signal transduction

Myelin-associated glycoprotein (MAG) is a potent inhibitor of neurite outgrowth from a variety of neurons. Here we show that gangliosides, GT1b and GD1a, as well as the Nogo receptor, are functional binding partners for soluble MAG-Fc. Postnatal cerebellar neurons from mice deficient in the GalNAcT gene are insensitive to MAG with regard to neurite outgrowth and lack in the activation of RhoA. MAG-Fc or the antibody to GT1b and GD1a elicits recruitment of p75(NTR.) to lipid rafts, specialized microdomain for signal transduction. Disruption of lipid rafts results in abolishment of inhibitory effects of MAG-Fc and the Nogo peptide. These findings establish gangliosides as functional binding partners for soluble MAG. Gangliosides may play a role in translocation of p75(NTR.) to lipid rafts for initiation of the signal transduction.     

Epigenetic factors up-regulate expression of myelin proteins in the dysmyelinating jimpy mutant mouse

Proteolipid protein (PLP) is a major structural component of central nervous system (CNS) myelin. Evidence exists that PLP or the related splice variant DM-20 protein may also play a role in early development of oligodendrocytes (OLs), the cells that form CNS myelin. There are several naturally occurring mutations of the PLP gene that have been used to study the roles of PLP both in myelination and in OL differentiation. The PLP mutation in the jimpy (jp) mouse has been extensively characterized. These mutants produce no detectable PLP and exhibit an almost total lack of CNS myelin. Additionally, most OLs in affected animals die prematurely, before producing myelin sheaths. We have studied cultures of jp CNS in order to understand whether OL survival and myelin formation require production of normal PLP. When grown in primary cultures, jp OLs mimic the relatively undifferentiated phenotype of jp OLs in vivo. They produce little myelin basic protein (MBP), never immunostain for PLP, and rarely elaborate myelin-like membranes. We report here that jp OLs grown in medium conditioned by normal astrocytes synthesize MBP and incorporate it into membrane expansions. Some jp OLs grown in this way stain with PLP antibodies, including an antibody to a peptide sequence specific for the mutant jp PLP. This study shows that: (1) an absence of PLP does not necessarily lead to dysmyelination or OL death; (2) OLs are capable of translating at least a portion of the predicted jp PLP; (3) the abnormal PLP made in the cultured jp cells is not toxic to OLs. These results also highlight the importance of environmental factors in controlling OL phenotype. © 1996 John Wiley & Sons, Inc.     

Regulation of neuronal nitric oxide synthase by histone,protamine, and myelin basic protein

We examined the effects of endogenous basic proteins rich in the amino acidL-arginine on neuronal NO synthase activity by monitoring cyclic GMP formation in intact neuron-like neuroblastoma N1E-115 cells. Histone, protamine and myelin basic protein significantly stimulated cyclic GMP formation, both in a time- and concentration-dependent manner. These effects were blocked by hemoglobin and NO synthase inhibitors. Removal of the extracellular/intracellular Ca²⁺ gradient by a Ca²⁺ chelator completely abolished the cyclic GMP responses elicited by histone and protamine, suggesting that influex of extracellular Ca²⁺ might be involved in their activation of NO synthase. The effects of myelin basic protein on cyclic GMP formation, however, appeared to be due to Ca²⁺ release from intracellular stores. In cytosolic preparations of rat cerebellum, these basic proteins inhibited the metabolism ofL-arginine intoL-citrulline by NO synthase. We conclude from our findings that endogenous basic proteins might be involved in the regulation of neuronal NO synthase activity. Their effects on the enzyme could be either stimulatory or inhibitory, depending on whether the basic proteins exert their effects extracellularly or intracellularly, respectively.