Age-Related Changes of Myelin Proteins in the Rat Peripheral Nervous System

Age-related changes in amounts of myelin proteins from rat sciatic nerve or spinal root were analyzed by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE). In the aged peripheral nerve myelin, the relative amounts of band 105K and proteins X and Y increased, whereas those of proteins P0 and P1 and band 190K decreased. Band 105K purified by preparative SDS-PAGE exhibited three bands of 105K, 28K, and 21K at the second electrophoresis. A repeated SDS-PAGE did not improve the purity of bank 105K, but increased the ratio of 21K to 28K. Compared with P0 protein, band 105K has a very similar peptide map pattern and amino acid composition, as well as the identical NH2 terminal residue, isoleucine. These findings suggest that band 105K is an aggregate form of P0 protein and its fragment, 21K. The 21K protein is a distinct entity from X protein. The quantitative and qualitative alterations in myelin proteins, as we report here, may reflect continuing demyelination and remyelination in aged peripheral nerves.     

Characterization of Basic Proteins from Goldfish Myelin

Abstract: Myelin basic protein (MBP) from common goldfish ( Carassius auratus ) myelin was extracted with dilute mineral acid. Immunological cross-reactivity of the goldfish MBP, with polyclonal antisera raised against bovine MBP, suggested that the goldfish protein has epitopes for these antibodies. It also reacted with a monoclonal antibody specific for a seven amino acid epitope (130–137) conserved in the MBP of most mammalian species. To characterize the charge heterogeneity of this protein, we iodinated the protein with ¹²⁵I and chromatographed it on a carboxymethyl cellulose-52 column together with a nonlabeled acid soluble fraction prepared from human white matter as a carrier protein. All of the goldfish protein was recovered in the unbound fraction, demonstrating that it was less cationic than the carrier protein (human MBP). We have also examined the urea alkaline gel profile of the goldfish MBP together with the human C-1, C-2, C-3, C-4, and C-8 components. The results from these experiments indicated that this MBP extracted from goldfish brain myelin lacked the microhet-erogeneity that is associated with MBPs from higher vertebrates. The MBPs from goldfish myelin were separated into their isoforms by reversed-phase HPLC. Amino acid compositions were determined for both the 17- and 14-kDa goldfish proteins. Amino acid analysis revealed similarities with the compositions of other MBPs; however, the serine content in both the 17- and 14-kDa proteins was higher than that of the human C-1, the mouse C-1 protein, and the shark proteins. The HPLC-purified 14-kDa goldfish protein was chemically cleaved with CNBr for partial sequence analysis. Even from the limited sequence obtained, the sequence ATAST was found in goldfish, which is also present in human, rabbit, and guinea pig MBPs.     

微管相关蛋白tau在不同年龄大鼠海马内的定位分布特点

目的 检测微管相关蛋白tau和Ser396/404位点磷酸化tau在胚胎期大鼠和出生后直至成熟期大鼠海马内的表达及变化规律,浅析与神经细胞分裂和分化的关系.方法 用免疫组织化学SABC法显示孕18d、出生后1d、1w、2w、2m的大鼠脑冠状切面总tau（R134d）、Ser396/404位点磷酸化tau（PHF-1）的表达.结果 ①胚胎期大鼠海马CA区的锥体细胞数量明显多于出生后,随着脑的发育,锥体细胞层的神经细胞数量逐渐下降;②孕18d大鼠海马内有丰富的总tau和PHF-1 tau的表达,并且海马内各区的阳性物质表达均匀,生后1d和1w两种物质表达仍然很强,阳性产物主要分布在海马CA1和CA2区以锥体细胞轴突为主要成分的始层,在锥体细胞树突集中的分子层和胞核内也有少量分布,而在生后2w和2m大鼠海马内各区均未见密集的阳性产物表达.结论 不同年龄大鼠海马内总tau和PHF-1 tau的表达和分布变化可能与神经系统发育过程中细胞的分裂和分化有关.     

Characterization and Biosynthesis of Cyclic-AMP-Binding Proteins in the Rat Central Nervous System

Cyclic-AMP-binding proteins in membrane and soluble fractions from rat forebrain were compared; membrane fractions included smooth and rough microsomes and a plasma membrane fraction enriched in synaptic membranes. Protein fractions were treated with 8-azido-[32P]cyclic AMP and ultraviolet irradiation to covalently tag cyclic-AMP-binding proteins. Labeled proteins were then analyzed by two-dimensional gel electrophoresis (2DGE) and fluorography. The soluble CNS proteins contained two major cyclic-AMP-binding species at 48K (48K 5.5 and 48K 5.45), differing slightly in their isoelectric points. Another protein was seen at 54K (54K 5.3) adjacent to the beta-tubulin subunits in the 2D electrophoretogram. The analysis of the smooth microsome and plasma membrane fractions differed from the soluble fraction in that there were two cyclic-AMP-binding proteins adjacent to the beta-tubulin region (54K 5.3 and 52K 5.3) differing slightly in apparent molecular weight. The membrane fractions also contained a cyclic-AMP-binding protein at 54K 5.8. The 52K 5.3 and 54K 5.8 species were unique to the membrane fractions. The rough microsomes did not contain detectable amounts of cyclic-AMP-binding proteins. Free polysomes were isolated from brain tissue, and translation products were analyzed by cyclic AMP affinity chromatography and immunopurification with antibodies to the brain specific type II regulatory subunit. The translation products that were found to bind cyclic AMP Sepharose are as follows: 48K 5.5, 48K 5.45, 52K 5.3, and 54K 5.8. These species comigrated with proteins that were photoaffinity-labeled in cytosol and membrane fractions.(ABSTRACT TRUNCATED AT 250 WORDS)     

POU蛋白调节中枢神经系统发育

POU蛋白是一组DNA特异的转录调节因子,属同源异形序列超家族.发育过程中,POU蛋白编码基因在中枢神经系统各部位的时空性表达决定神经细胞的发育与分化.     

MAPping out distribution routes for kinesin couriers

In the crowded environment of eukaryotic cells, diffusion is an inefficient distribution mechanism for cellular components. Long‐distance active transport is required and is performed by molecular motors including kinesins. Furthermore, in highly polarised, compartmentalised and plastic cells such as neurons, regulatory mechanisms are required to ensure appropriate spatio‐temporal delivery of neuronal components. The kinesin machinery has diversified into a large number of kinesin motor proteins as well as adaptor proteins that are associated with subsets of cargo. However, many mechanisms contribute to the correct delivery of these cargos to their target domains. One mechanism is through motor recognition of sub‐domain‐specific microtubule (MT) tracks, sign‐posted by different tubulin isoforms, tubulin post‐translational modifications, tubulin GTPase activity and MT‐associated proteins (MAPs). With neurons as a model system, a critical review of these regulatory mechanisms is presented here, with a particular focus on the emerging contribution of compartmentalised MAPs. Overall, we conclude that – especially for axonal cargo – alterations to the MT track can influence transport, although in vivo, it is likely that multiple track‐based effects act synergistically to ensure accurate cargo distribution.     

Production and Characterization of Monoclonal Antibodies to Peripheral and Central Nervous System Myelin

Monoclonal antibodies against P0, myelin basic protein, or myelin-associated glycoprotein were generated by fusing mouse myeloma cells with spleen cells from BALB/c mice immunized with central and peripheral nervous system myelin proteins. The antibodies secreted were either IgG, IgM, or IgA. Clone C6B5 (iso-type IgM) secreted antibody(ies) that bound to both myelin basic protein and myelin-associated glycoprotein, although binding of antibody to myelin basic protein as detected by the immunoblot technique appeared to be much less than to the myelin-associated glycoprotein. Antibodies were characterized in solid-phase radioimmunoassay for their species cross-reaction, and histologically for the specificity of binding to myelin in central and peripheral nervous system tissues. These monoclonal reagents should prove valuable in studying CSF and myelin-producing cells, since in both cases the concentration of myelin proteins is low.     

Analysis of α-Bungarotoxin Binding in the Goldfish Central Nervous System

Abstract: We report here the equilibrium, kinetic, and pharmacological analysis of α-¹²⁵I-bungarotoxin (α-¹²⁵I-Bgt) binding to a Triton x-100-solubilized goldfish brain synaptosomal fraction. In addition, a refined analysis of equilibrium binding to a particulate synaptosomal fraction is presented. Equilibrium binding from both particulate and soluble fractions revealed an apparent heterogeneity of binding sites. Kinetic analysis of the soluble receptor revealed linear association kinetics and nonlinear dissociation kinetics. The dissociation curve suggested the presence of at least two rate constants. Potential sources of the binding heterogeneity found in both the equilibrium binding and dissociation kinetics experiments are (1) multiple receptor species, (2) multiple ligand species, and (3) different, possibly interconvertible, states of a single receptor type. No evidence for the first two alternatives was found. Support for the third alternative was obtained by observing the effect of cholinergic ligands on α-¹²⁵I-Bgt dissociation. Carbamylcholine and d -tubocurarine increased the apparent proportion of rapidly dissociating sites, suggesting that the two binding affinities can be interconverted and may arise from a single receptor type. Evidence concerning the identity of the α -Bgt binding protein as a nicotinic acetylcholine receptor is discussed.     

Characterization and Regional Distribution of Individual Gangliosides in Goldfish Central Nervous System

Gangliosides were partially purified from goldfish brain and fractionated by DEAE Fractogel column chromatography. Each fraction was then analyzed by HPTLC and also by HPLC after conversion of the gangliosides to 2,4-dinitrophenylhydrazides. The tetrasialoganglioside GQ1c was found to constitute more than 50% of the total gangliosides. Gangliosides in smaller quantities were also tentatively identified. These included GT1b, GT1c, GT2, GT3, GD1a, and several others. By using this information, the amounts of individual gangliosides in various regions of goldfish central nervous system were compared. Although all areas of brain examined contained similar concentrations of gangliosides, with GQ1c as the predominant component, retina and optic nerve contained significantly lower concentrations of GQ1c, and GM3 was the major component.     

Abnormal Expression of the Myelin-Associated Glycoprotein in the Central Nervous System of Dysmyelinating Mutant Mice

Total cytoplasmic brain RNA was isolated at two different ages from three neurological mutant mice (qk/qk, jp/Y, and shi/shi) and their apparently normal littermates. This RNA was translated in vitro in a rabbit reticulocyte lysate system. Myelin-associated glycoprotein (MAG)-related polypeptides were immunoprecipitated from equal amounts of total translation products derived from mRNA of mutant animals, normal littermates, or control animals. The developmentally regulated synthesis of MAG polypeptides was compared among the mutants and normal animals. mRNA from qk/qk brains synthesized an overabundance of p67MAG (five- to sevenfold) which may be compensation for a decreased synthesis of p72MAG. mRNA from jp/Y brains synthesized less than 10% of normal amounts of both MAG polypeptides. The quantity of MAG synthesized by 15-day shi/shi brain mRNA was slightly decreased compared with normal brain mRNA but the quantity of MAG synthesized by adult shi/shi brain mRNA was normal. No apparent differences were detected in the sizes of the MAG polypeptides synthesized by any of the mutants studied. The data suggest that the genetic defect in qk/qk mutants directly or indirectly affects the coordinated developmental regulation of MAG polypeptide synthesis leading to an overabundance of the MAG polypeptide that is normally found in older animals. The jp/Y mutation appears to affect general myelin protein synthesis. Finally, shi/shi mutants may have a delayed synthesis of MAG. The data are discussed in the light of recent observations concerning the synthesis of myelin proteins and their proposed role in myelin assembly.     

Participation of Septin Cytoskeletal Proteins in the Nervous System Functioning

Septins, the cytoskeletal proteins discovered in the 1970s in budding yeast cells, are currently detected in most postmitotic cells of animals and humans. In the last decade, significant progress has been made in understanding the biochemical properties of septins and their biological functions. An increasing number of studies show that these proteins play an important role in the development and physiology of specific tissues and organs. The review surveys classification, major functions and localization of septins in the nervous system of mammals and humans. Models describing the mechanisms of the septin involvement in the neurotransmitter secretion from nerve endings and the role of septins in the pathogenesis of various neurodegenerative diseases are discussed.     

The Association of Tau-Like Proteins with Vimentin Filaments in Cultured Cells

There is increasing evidence that the different polymers that constitute the cytoskeleton are interconnected to form a three-dimensional network. The macromolecular interaction patterns that stabilize this network and its intrinsic dynamics are the basis for numerous cellular processes. Within this context,in vitrostudies have pointed to the existence of specific associations between microtubules, microfilaments, and intermediate filaments. It has also been postulated that microtubule-associated proteins (MAPs) are directly involved in mediating these interactions. The interactions of tau with vimentin filaments, and its relationships with other filaments of the cytoskeletal network, were analyzed in SW-13 adenocarcinoma cells, through an integrated approach that included biochemical and immunological studies. This cell line has the advantage of presenting a wild-type clone (vim+) and a mutant clone (vim−) which is deficient in vimentin expression. We analyzed the cellular roles of tau, focusing on its interactions with vimentin filaments, within the context of its functional aspects in the organization of the cytoskeletal network. Cosedimentation experiments of microtubular protein with vimentin in cell extracts enriched in intermediate filaments, combined with studies on the direct interaction of tau with nitrocellulose-bound vimentin and analysis of tau binding to vimentin immobilized in single-strand DNA affinity columns, indicate that tau interacts with the vimentin network. These studies were confirmed by a quantitative analysis of the immunofluorescence patterns of cytoskeleton-associated tubulin, tau, and vimentin using flow cytometry. In this regard, a decrease in the levels of tau associated to the cytoskeletal network in the vim− cell mutant compared with the wild-type clones was observed. However, immunofluorescence data on SW-13 cells suggest that the absence of a structured network of vimentin in the mutant vim− cells does not affect the cytoplasmic organization formed by microtubules and actin filaments, when compared with the wild-type vim+ cells. These studies suggest that tau associates with vimentin filaments and that these interactions may play a structural role in cells containing these filaments.     

Release of Proteins from the Surface of Bovine Central Nervous System Myelin by Salts and Phospholipases

Incubation of bovine CNS myelin with phospholipase C from Bacillus cereus under conditions that lead to extensive phospholipid degradation caused 10% of the myelin protein to be released from the membrane. The myelin basic protein (MBP) was a major component of the dissolved protein. Comparable incubations with phospholipase C from Clostridium perfringens, phosphatidylinositol-specific phospholipase C from Staphylococcus aureus, or cabbage phospholipase D removed little MBP. However, concentrations of sodium chloride near 1 M and concentrations of divalent metal ions between 50 and 600 mM released typically 9-12% of the total myelin protein, with MBP again as the predominant component. Repetitive washing with calcium chloride solutions resulted in dissolution of over 90% of the MBP. When myelin was incubated in 1.0 M sodium chloride or 25 mM calcium chloride, the MBP was cleaved largely into two major peptides with apparent molecular weights near 14,000 and 12,000, but with 200 mM or higher concentrations of calcium chloride most of this protein remained intact. With appropriate manipulation of the ionic milieu, it is thus possible to remove most of this extrinsic protein from the myelin surface under relatively mild conditions. The conditions that release the protein suggest that it is held at the membrane surface by ionic interactions.     

Biochemical Demonstration of the Myelin-Associated Glycoprotein in the Peripheral Nervous System

Abstract: Recent immunocytochemical studies indicated that the myelin-associated glycoprotein (MAG) is localized in the periaxonal region of central nervous system (CNS) and peripheral nervous system (PNS) myelin sheaths but previous biochemical studies had not demonstrated the presence of MAG in peripheral nerve. The glycoproteins in rat sciatic nerves were heavily labeled by injection of [³H]fucose in order to re-examine whether MAG could be detected chemically in peripheral nerve. Myelin and a myelin-related fraction, WI, were isolated from the nerves. Labeled glycoproteins in the PNS fractions were extracted by the lithium diiodosalicylate (LIS)-phenol procedure, and the extracts were treated with antiserum prepared to CNS MAG in a double antibody precipitation. This resulted in the immune precipitation of a single [³H]fucose-labeled glycoprotein with electrophoretic mobility very similar to that of [¹⁴C]fucose-labeled MAG from rat brain. A sensitive peptide mapping procedure involving iodination with Bolton-Hunter reagent and autoradiography was used to compare the peptide maps generated by limited proteolysis from this PNS component and CNS MAG. The peptide maps produced by three distinct proteases were virtually identical for the two glycoproteins, showing that the PNS glycoprotein is MAG. The MAG in the PNS myelin and Wl fractions was also demonstrated by Coomassie blue and periodic acid-Schiff staining of gels on which the whole US-phenol extracts were electrophoresed, and densitometric scanning of the gels indicated that both fractions contained substantially less MAG than purified rat brain myelin. The presence of MAG in the periaxonal region of both peripheral and central myelin sheaths is consistent with a similar involvement of this glycoprotein in axon-sheath cell interactions in the PNS and CNS.     

Acetylation/deacetylation and microtubule associated proteins influence flagellar axonemal stability and sperm motility

PTMs and microtubule-associated proteins (MAPs) are known to regulate microtubule dynamicity in somatic cells. Reported literature on modulation of α-tubulin acetyl transferase (αTAT1) and histone deacetylase 6 (HDAC6) in animal models and cell lines illustrate disparity in correlating tubulin acetylation status with stability of MT. Our earlier studies showed reduced acetyl tubulin in sperm of asthenozoospermic individuals. Our studies on rat sperm showed that on inhibition of HDAC6 activity, although tubulin acetylation increased, sperm motility was reduced. Studies were therefore undertaken to investigate the influence of tubulin acetylation/deacetylation on MT dynamicity in sperm flagella using rat and human sperm. Our data on rat sperm revealed that HDAC6 specific inhibitor Tubastatin A (T) inhibited sperm motility and neutralized the depolymerizing and motility debilitating effect of Nocodazole. The effect on polymerization was further confirmed in vitro using pure MT and recHDAC6. Also polymerized axoneme was less in sperm of asthenozoosperm compared to normozoosperm. Deacetylase activity was reduced in sperm lysates and axonemes exposed to T and N+T but not in axonemes of sperm treated similarly suggesting that HDAC6 is associated with sperm axonemes or MT. Deacetylase activity was less in asthenozoosperm. Intriguingly, the expression of MDP3 physiologically known to bind to HDAC6 and inhibit its deacetylase activity remained unchanged. However, expression of acetyl α-tubulin, HDAC6 and microtubule stabilizing protein SAXO1 was less in asthenozoosperm. These observations suggest that MAPs and threshold levels of MT acetylation/deacetylation are important for MT dynamicity in sperm and may play a role in regulating sperm motility.     

Role of Matricellular Proteins in Disorders of the Central Nervous System

Matricellular proteins (MCPs) are actively expressed non-structural proteins present in the extracellular matrix, which rapidly turnover and possess regulatory roles, as well as mediate cell–cell interactions. MCPs characteristically contain binding sites for other extracellular proteins, cell surface receptors, growth factors, cytokines and proteases, that provide structural support for surrounding cells. MCPs are present in most organs, including brain, and play a major role in cell–cell interactions and tissue repair. Among the MCPs found in brain include thrombospondin-1/2, secreted protein acidic and rich in cysteine family (SPARC), including Hevin/SC1, Tenascin C and CYR61/Connective Tissue Growth Factor/Nov family of proteins, glypicans, galectins, plasminogen activator inhibitor (PAI-1), autotaxin, fibulin and perisostin. This review summarizes the potential role of MCPs in the pathogenesis of major neurological disorders, including Alzheimer’s disease, amyotrophic lateral sclerosis, ischemia, trauma, hepatic encephalopathy, Down’s syndrome, autism, multiple sclerosis, brain neoplasms, Parkinson’s disease and epilepsy. Potential therapeutic opportunities of MCP’s for these disorders are also considered in this review.     

Stimulation of Tubulin-Dependent ATPase Activity in Microtubule Proteins from Porcine Brain by Taxol

Taxol, an antimitotic agent that induces microtubule assembly, stimulated tubulin-dependent Mg2+-ATPase activity of microtubule-associated proteins (MAPs). A concentration-dependent increase in the rate of ATP hydrolysis was observed. Taxol acted through its binding to the tubulin molecule on MAP ATPase, and maximal stimulation, which was found at approximately equal concentrations of taxol and tubulin, reached about 140% of the original level in the absence of taxol. Taxol enhanced ATP hydrolysis by a mixture of MAPs and tubulin, and this continued at a steady linear rate even when the polymerization had approached a plateau. In the presence of taxol, a large portion of ATPase activity and protein was recovered in the pellet after centrifugation at 70,000 g for 60 min at 25 degrees C. Both colchicine and podophyllotoxin inhibited taxol-stimulated ATPase activity via the same mechanism by which they inhibited taxol-induced microtubule polymerization. The stimulation by taxol was not found in the presence of Ca2+ alone but required Mg2+. We conclude that tubulin effectively stimulates Mg2+-ATPase activity of MAPs under conditions that induce tubulin polymerization.