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.     

Effects of Exogenous Hexacosanoic Acid on Biochemical Myelin Composition in Weaning and Post-Weaning Rats

X-linked Adrenoleukodistrophy (ALD) is characterized by an increase of very long chain fatty acids (VLCFA) in particular of hexacosanoic acid (HA), in tissues and fluids. The biochemical abnormality is due to the dysfunction of peroxisomal degradation of VLCFA. To-date it is unclear if the demyelination which characterizes this disease is the direct consequence of HA accumulation. In order to investigate whether the large amounts of exogenous HA could affect myelin synthesis, 500 g of this fatty acid dissolved in peanut oil were administered daily and by gavage to newborn rats. Since myelin is actively synthesized during early neonatal life and it can be altered by en-vironmental factors including diet, we analyzed lipid and protein composition of myelin after 20, 30 and 60 days of HA administration. Our results show that exogenous HA is incorporated in myelin where it determines biochemical alterations in normal rats having a functioning peroxisomal system. Even though the differences between controls and treated rats are slight, we observed in test rats, a decrease of 23-cyclic nucleotide 3-phosphohydrolase (CNPase) activity and of myelin basic protein (MBP) content at any time studied. The decrease of glycolipids (GL) was present only after 20 days of treatment. Since these parameters are related to myelin development, our data lead us to think that the myelin of the treated animals is less mature than that of controls.     

The regional and subcellular distribution of calcium activated neutral proteinase (CANP) in the bovine central nervous system

Calcium-activated neutral proteinase (CANP) activity was determined in subcellular fractions and in different regions of bovine brain. The CANP specific activity in spinal cord and corpus callosum, areas rich in myelin, were almost six-fold greater than cerebral cortex and cerebellum. Treatment of whole homogenate and myelin with 0.1% Triton X-100 increased the CANP activity by tenfold. Subcellular fractions were prepared from bovine brain gray and white matter. Most of the CANP activity (70%) was in the primary particulate fractions P₁ (nuclear), P₂ (mitochondrial) and P₃ (microsomal). On subfractionation of each particulate fraction, the majority of the activity (greater than 50%) was recovered in the myelin-enriched fractions (P₁A, P₂A, P₃A) which separate at the interphase of 0.32 M- and 0l85 M-sucrose. The distribution of activity was P₂A>P₁A>P₃A. Further purification of myelin (of P₂A) increased the specific activity over homogenate by more than three-fold. The same myelin fractions contained the highest proportion (60%) and specific activity (five-fold increase) of CNPase. The enzyme activity in different regions of brain and in subcellular fractions was increased by 20–39% after the inhibitor was removed. Electron microscopic study confirmed that the myelin fractions were highly purified. The cytosolic fraction contained 20–30% of the total homogenate CANP activity. Other fractions contained low enzyme activity. CANP was identified in the purified myelin fraction by electroimmublot-technique. It is concluded that the bulk of CANP in CNS is tightly bound to the membrane, may be masked or hidden and is intimately associated with the myelin sheath.Abbreviations Used CANP calcium-activated neutral proteinase - CNPase adenosine-2, 3-cyclic nucleotide 3-phosphohydrolase     

The presence of 17K Mr protein,a major specific substrate for kinase C,found in the Triton-insoluble fraction of synaptosome prepared from rat brain

Cytoskeletal preparation obtained from synaptosome fractions of rat cerebrum contained the activity of kinase C, which phosphorylated 17K Mr protein endogenous to the preparation. The kinase C activity associated with the synaptosome cytoskeletons is greater in the cerebellum and hippocampus than in the cerebrum. The enhancement rates of phosphorylation of the 17K Mr protein were 293%, 544%, and 526% in the Triton X-100-insoluble fractions of synaptosomes prepared from cerebral cortex, hippocampus, and cerebellum, respectively. The 17K Mr protein was distinct from myelin basic protein (MBP) for the following reasons: 1) The electrophoretic mobility of the protein was slightly smaller than that of major MBP of rat in the polyacrylamide gel of 10–20% linear gradient, and the protein was not contained in the purified rat myelin. 2) The isoelectric point of the protein was in neutral range, whereas that of MBP was in alkaline one. 3) The 17K Mr protein did not cross-react with anti-MBP antibody. The protein was shown to be a major substrate contained in the cytoskeletal preparation of synaptosome obtained from cerebrum except for contaminating MBP. Only serine residue of the 17K Mr protein was phosphorylated by the kinase C endogenous to the preparation. The results suggest strongly that the synaptic role of protein kinase C through phosphorylation of the 17K Mr protein.Abbreviations used EGTA ethyleneglycol-bis(-aminoethyl ether) - HEPES N-2-hydroxyethyl-piperazine-N-2-ethanesulfonic acid - MBP myelin basic protein - SDS sodium dodecyl sulfate - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis - SPM synaptic plasma membrane     

Generation of a monoclonal antibody against the myelin protein CNP (2′,3′-cyclic nucleotide 3′-phosphodiesterase) suitable for biochemical and for immunohistochemical investigations of CNP

The functional role of CNP (2,3-cyclic nucleotide 3-phosphodiesterase), a minor component of central and peripheral myelin is still unclear. Here we describe preparation of a monoclonal antibody directed against CNP. The antibody, of the immunoglobulin IgG₁ type, raised with a basic 46 kDa membrane-associated protein solubilized from pig cerebellar membranes, can be used to detect immunoreactivity in solubilized brain homogenates from pig, mouse, rat, sheep, cow and man, in cerebrum and cerebellum, but not in other tissues such as liver, skeletal and heart muscle. The antibody recognizes the CNP doublet band and shows no cross-reactivity with any of the other brain proteins solubilized. In tissue sections from paraformaldehyde-fixed rat brain the antigen was localized in oligodendrocytes. In cultured glial cells from newborn mice the antibody stained cells which were identified as oligodendrocytes by co-localization of myelin basic protein. Even cells from a C6 rat glioma cell line, which contain very little of CNP, were labeled by the monoclonal antibody. Thus the monoclonal antibody recognizing CNP from several species is suitable for immunocytochemical investigations and also for biochemical studies of CNP, since the antibody has been employed for immunoprecipitation and immunopurification of CNP in crude brain homogenates.     

Activities of myelin bound cytidine 5′-diphosphate-choline 1, 2 diacyl-glycerol choline phosphotransferase and uridine 5′-diphosphate-galactose-ceramide galactosyltransferase under restricted food intake

Activities of cytidine 5′-diphosphate-choline glycerol choline phosphotransferase and uridine 5′-diphosphate galactose-ceramide galactosyltransferase were determined in isolated myelin in different brain regions of control, and rats with restricted food intake. Kinetic experiments indicated an increase inK _m value of phosphocholinetransferase in brain stem of undernourished rats, without significant change in the specific activity of this enzyme. Stimulation of this myelin bound enzyme activity was also evident in the animals when myelin was treated with the detergent: Tween CF. 54. Though specific activities of galactosyl transferase in myelin of undernourished rats were significantly diminished, theK _m of this enzyme was unaltered. These studies point to an adverse effect of early nutritional stress on the activities of enzymes bound to myelin membrane which has hitherto been considered metabolically inert.     

Yeast surviving factor Svf1 as a new interacting partner, regulator and in vitro substrate of protein kinase CK2

Since Svf1 is phosphoprotein, we investigated whether it was a substrate for protein kinase CK2. According to the amino acid sequence Svf1 harbours 20 putative CK2 phosphorylation sites. Here, we have reported cloning, overexpression, purification and characterization of yeast Svf1 as a substrate for three forms of yeast CK2. Svf1 serves as a substrate for both the recombinant CK2α (K _m 0.35 μM) and CK2α′ (K _m 0.18 μM) as well as CK2 holoenzyme (K _m 1.1 μM). Different K _m values argue that CK2β(β′) subunit has an inhibitory effect on the activity of both CK2α and CK2α′ towards surviving factor Svf1. Reconstitution of α′₂ββ′ isoform of CK2 holoenzyme shows that β/β′ subunits have regulatory effect depending on the kind of CK2 catalytic subunit. This effect was not observed in the case of α₂ββ′ isoform, which may be due to interaction between Svf1 and regulatory CK2β subunit (shown by co-immunoprecipitation experiments). Interactions between CK2 subunits and Svf1 protein may have influence on ATP as well as ATP-competitive inhibitors (TBBt and TBBz) binding. CK2 phosphorylates up to six serine residues in highly acidic peptide K¹⁹⁹EVIPESDEEESSADEDDNEDEDEESGDSEEESGSEEESDSEEVEITYED²⁴⁸ of the Svf1 protein in vitro. Presented data may help to elucidate the role of protein kinase CK2 and Svf1 in the regulation of cell survival pathways.     

Hoxd1 is Expressed by Oligodendroglial Cells and Binds to a Region of the Human Myelin Oligodendrocyte Glycoprotein Promoter in vitro

(1) Little information exists on the role of clustered Hox genes in oligodendrocyte (OG) development. This study examines the expression profile of Hoxd1 and identifies a potential downstream target in the OG lineage. (2) Immunocytochemical analysis of primary mixed glial cultures demonstrated Hoxd1 was expressed throughout OG development. (3) A human myelin protein gene, myelin oligodendrocyte glycoprotein (MOG), was identified as a putative downstream target of Hoxd1 through Genbank searches utilizing the Hoxd1 homeodomain consensus binding sequence. (4) The dissociation coefficient constant (K _D) and dissociation rate constant (k _d) of the Hoxd1–MOG complex, determined using electrophoretic mobility shift assays (EMSAs), were estimated to be 1.9 × 10⁻⁷ M and 1.3 × 10⁻³ s⁻¹, respectively. The DNA–Hoxd1 homeodomain complex had a half-life (t _1/2) of 15 min. (5) Mutational analysis of Hoxd1–MOG complexes revealed the binding affinity of M1 (with mutation from ⁻¹⁰⁵⁴5′-TAAT-3′⁻¹⁰⁵¹ to TACT within the consensus binding site) and M2 (with mutation from ^-10545′-TAATTG-3′^-1049 to TAATCC within the consensus binding site) probes to the MOG promoter was severely affected. Thus the TAATTG core of the binding sequence appears important for Hoxd1 specificity. (6) Analysis of the involvement of TAAT sites adjacent to the consensus sequence in Hoxd1 binding showed the binding affinity of the M3 probe was affected, but not as severely as the M1 and M2 probes. These in vitro results suggest the TTTAATTGTA sequence is involved in Hoxd1 binding to the MOG promoter but neighboring TAAT sites may also be needed. Thus, MOG may be a target of Hoxd1.     

Localization of 5′-nucleotidase in bovine brain myelin fraction and myelin subfractions

Purified myelin from fresh calf brain white matter was subfractionated in a discontinuous sucrose gradient; significant recovery of protein and 2,3-cyclic nucleotide 3-phosphohydrolase (CNP) and 5-nucleotidase (5N) activities occurred in all three obtained subfractions, the highest recovery being in the light subfraction; highest 5N and CNP specific activities were in medium myelin. Purified myelin was also subfractionated in a continuous sucrose gradient, with a similar localization of protein; CNP activity and 5N activity maxima suggest that myelin may be a predominant locus of 5N in bovine brain white matter. Freezing of brain white matter caused an increase in protein and in CNP and 5N total activity recoveries in denser myelin subfractions. Cytochemistry showed the reaction product of 5N in the whole myelin fraction to be associated with the innermost, outermost and medial compact myelin layers. Effects of non-ionic detergent (Lubrol WX) on 5N activity were studied, and the results also suggest the intrinsic nature of 5N as an ectoenzyme in myelin membranes. Lubrol WX was viewed as an advisable detergent for the stimulation of myelin 5N activity, but not for the solubilization of this enzyme.     

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)     

Study of myelin purity in relation to axonal contaminants

Axonal remnants are considered a probable source of contamination of isolated myelin in view of the relatively tight axon-glial intercellular junction. Using the rabbit optic system to label specifically axonal components, we have found the levels of such contaminants to depend on the myelin isolation procedure, the tissue source, and the nature of the contaminant. A procedure employing repetitive treatments with EGTA was found to be highly effective in removing proline-labeled axonal proteins, the estimated upper limit of such contamination being approximately 0.6–1.2% of the myelin protein. The standard isolation procedure of Norton and Poduslo, supplemented with an additional discontinuous gradient step, proved equally effective in removing rapidly transported proteins from myelin isolated from the superior colliculus or lateral geniculate body. When the optic tract was the source, however, the EGTA procedure proved more effective in removing both rapidly and slowly transported proteins. Axonal gangliosides labeled with N-[ ³ H]acetylmannosamine were efficiently removed by both procedures, adding support to the proposition that gangliosides detected in isolated myelin are intrinsic to that membrane.     

Partial deficiencies in the myelin proteins of developing mice heterozygous for the shiverer mutation

The central nervous system of the shiverer mouse is known to be severely deficient in myelin. Animals heterozygous for this autosomal-recessive mutation were crossed, and the myelin proteins were examined in the brains and spinal cords of shiverers and unaffected littermates among the offspring. In the brains and spinal cords of nine of the 14 unaffected littermates examined, the quantities of the myelin basic and proteolipid proteins were lower than normal. Furthermore, in the brains of heterozygotes 33 to ～ 150 days old, the myelin basic and proteolipid proteins were reduced in amount, compared to wild-type controls; the myelin basic protein was also present in subnormal amounts in the spinal cords from heterozygous animals at the ages of 17 to 150 days. More severe reductions in the quantities of the myelin proteins were observed in central nervous system tissue from homozygous shiverer mice, and the quantity of the myelin proteolipid protein in the central nervous system of the shiverer mouse, expressed as a ratio to the control value at each age, underwent a developmental decline. In heterozygotes, as well as shiverers, the peripheral nerves were also deficient in the P₁ and P_r proteins, which are the same as the basic proteins in rodent central nervous system myelin. The findings regarding heterozygotes suggest that the defective primary gene product in the shiverer mouse could be the myelin basic protein itself or a protein required for a rate-limiting step in the processing of the myelin basic protein.     

Thyroid Hormones Reorganize the Cytoskeleton of Glial Cells Through Gfap Phosphorylation and Rhoa-Dependent Mechanisms

Thyroid hormones (3,5,3′-triiodo-l-thyronine, T₃; 3,5,3′,5′-l-tetraiodothyronine, T₄; TH) play crucial roles in the growth and differentiation of the central nervous system. In this study, we investigated the actions of TH on proliferation, viability, cell morphology, in vitro phosphorylation of glial fibrillary acidic protein (GFAP) and actin reorganization in C6 glioma cells. We first observe that long-term exposure to TH stimulates cell proliferation without induce cell death. We also demonstrate that after 3, 6, 12, 18, and 24 h treatment with TH, C6 cells and cortical astrocytes show a process-bearing shape. Furthermore, immunocytochemistry with anti-actin and anti-GFAP antibodies reveals that TH induces reorganization of actin and GFAP cytoskeleton. We also observe an increased in vitro ³²P incorporation into GFAP recovered into the high-salt Triton insoluble cytoskeletal fraction after 3 and 24 h exposure to 5×10⁻⁸ and 10⁻⁶ M T₃, and only after 24 h exposure to 10⁻⁹ M T₄. These results show a T₃ action on the phosphorylating system associated to GFAP and suggest a T₃-independent effect of T₄ on this cytoskeletal protein. In addition, C6 cells and astrocytes treated with lysophosphatidic acid, an upstream activator of the RhoA GTPase pathway, totally prevented the morphological alterations induced by TH, indicating that this effect could be mediated by the RhoA signaling pathway. Considering that IF network can be regulated by phosphorylation leading to reorganization of IF filamentous structure and that alterations of the microfilament organization may have important implications in glial functions, the effects of TH on glial cell cytoskeleton could be implicated in essential neural events such as brain development.     

Topology of wolfgram proteins and 2′, 3′-cyclic nucleotide 3′-phosphodiesterase in CNS myelin: Studies with proteases

The topological disposition of Wolfgram proteins (WP) and their relationship with 2, 3-cyclic nucleotide 3-phosphodiesterase (CNPase) in human, rat, sheep, bovine, guinea pig and chicken CNS myelin was investigated. Controlled digestion of myelin with trypsin gave a 35KDa protein band (WP-t) when electrophoresed on dodecyl sulfate-polyacrylamide gel in all species. Western blot analysis showed that the WP-t was derived from WP. WP-t was also formed when rat myelin was treated with other proteases such as kallikrein, thermolysin and leucine aminopeptidase. Staining for CNPase activity on nitrocellulose blots showed that WP-t is enzymatically active. Much of the CNPase activity remained with the membrane fraction even after treatment with high concentrations of trypsin when WP were completely hydrolysed and no protein bands with M.W>14KDa were detected on the gels. Therefore protein fragments of WP with M.W<14KDa may contain CNPase activity. From these results, it is suggested that the topological disposition of all the various WP is such that a 35KDa fragment is embedded in the lipid bilayer and the remaining fragment exposed at the intraperiod line in the myelin structure which may play a role in the initiation of myelinogenesis.     

In vivo phosphorylation of 2′,3′-cyclic nucleotide 3′-phosphohydrolase (CNP): CNP in brain myelin is phosphorylated by forskolin- and phorbol ester-sensitive protein kinases

2,3-cyclic nucleotide 3-phosphohydrolase (CNP) was phosphorylated in vivo, in brain slices and in a cell free system. Phosphoamino acid analysis of immunoprecipitated CNP labeled in vivo and in brain slices revealed phosphorylation of phosphoserine (94%) and phosphothreonine (5%) residues. Phosphorylation of CNP increased by 3-fold after brain slices were incubated with forskolin. Similarly, incubation of isolated myelin with [-³²]ATP with cAMP (5 M) and cAMP (5 M) + catalytic unit of cAMP dependent protein kinase dramatically increased CNP2 phosphorylation by 4- and 6-fold, respectively. It is feasible that CNP2 was predominantly phosphorylated on serine and/or threonine residues of the amino terminal peptide of CNP2, and this phosphorylation was catalyzed by protein kinase A. Phosphorylation of CNP1 and CNP2 increased 2-fold by incubating brain slices with phorbol ester. Forskolin and phorbol ester increased the phosphorylation of single, but distinct, CNP peptides. We present the first biochemical evidence that CNP2, on a protein mass basis, is far more heavily phosphorylated than CNP1, suggesting there are more phosphorylation sites on CNP2 than CNP1 and that at least one site is located on the 20-amino acid terminus of CNP2 and that is is likely a PKA site.     

Phosphorylation is Involved in the Regulation of the Taurine Influx Via the β-system in Ehrlich Ascites Tumor Cells

The role of 3′,5′-cyclic adenosine monophosphate (cAMP), protein kinase A (PKA), protein kinase C (PKC) and phosphatases in the regulation of the taurine influx via the β-system in Ehrlich ascites tumor cells has been investigated. The taurine uptake by the β-system in Ehrlich cells is inhibited when PKC is activated by phorbol 12-myristate 13-acetate (PMA) and when protein phosphatases are inhibited by calyculin A (CLA). On the other hand, taurine uptake by the β-system is stimulated by an increased level of cAMP or following addition of N⁶,2′-O-dibutyryl-3′,5′-cyclic adenosine monophosphate (dbcAMP). The effect of dbcAMP is partially blocked by addition of the protein kinase inhibitor H-89, and suppressed in the presence of CLA. It is proposed that the β-system in the Ehrlich cells exists in three states of activity: State I, where a PKC phosphorylation site on the transporter or on a regulator is phosphorylated and transport activity is low. State II, where the PKC phosphorylation site is dephosphorylated and transport activity is normal. State III, representing a state with high transport activity, induced by an elevated cellular cAMP level. Apparently, cAMP preferentially stimulates taurine transport when the β-system is in State II. Received: 8 September/Revised: 9 November 1995     

UTP and ATP increase extracellular signal-regulated kinase 1/2 phosphorylation in bovine chromaffin cells through epidermal growth factor receptor transactivation

Adenosine triphosphate (ATP) is coreleased with catecholamines from adrenal medullary chromaffin cells in response to sympathetic nervous system stimulation and may regulate these cells in an autocrine or paracrine manner. Increases in extracellular signal-regulated kinase (ERK) 1/2 phosphorylation were observed in response to ATP stimulation of bovine chromaffin cells. The signaling pathway involved in ATP-mediated ERK1/2 phosphorylation was investigated via Western blot analysis. ATP and uridine 5′-triphosphate (UTP) increased ERK1/2 phosphorylation potently, peaking between 5 and 15 min. The mitogen-activated protein kinase (MAPK/ERK)-activating kinase (MEK) inhibitor PD98059 blocked this response. UTP, which is selective for G-protein-coupled P2Y receptors, was the most potent agonist among several nucleotides tested. Adenosine 5′-O-(3-thio) triphosphate (ATPγS) and ATP were also potent agonists, characteristic of the P2Y₂ or P2Y₄ receptor subtypes, whereas agonists selective for P2X receptors or other P2Y receptor subtypes were weakly effective. The receptor involved was further characterized by the nonspecific P2 antagonists suramin and reactive blue 2, which each partially inhibited ATP-mediated ERK1/2 phosphorylation. Inhibitors of protein kinase C (PKC), protein kinase A (PKA), Ca²⁺/calmodulin-dependent protein kinase II (CaMKII), and phosphoinositide-3 kinase (PI3K) had no effect on ATP-mediated ERK1/2 phosphorylation. The Src inhibitor PP2, epidermal growth factor receptor (EGFR) inhibitor AG1478, and metalloproteinase inhibitor GM6001 decreased ATP-mediated ERK1/2 phosphorylation. These results suggest nucleotide-mediated ERK1/2 phosphorylation is mediated by a P2Y₂ or P2Y₄ receptor, which stimulates metalloproteinase-dependent transactivation of the EGFR.     

May Cyclic Nucleotides Be a Source for Abiotic RNA Synthesis?

Nucleic bases are obtained by heating formamide in the presence of various catalysts. Formamide chemistry also allows the formation of acyclonucleosides and the phosphorylation of nucleosides in every possible position, also affording 2′,3′ and 3′,5′ cyclic forms. We have reported that 3′,5′ cyclic GMP and 3′,5′ cyclic AMP polymerize in abiotic conditions yielding short oligonucleotides. The characterization of this reaction is being pursued, several of its parameters have been determined and experimental caveats are reported. The yield of non-enzymatic polymerization of cyclic purine nucleotides is very low. Polymerization is strongly enhanced by the presence of base-complementary RNA sequences.     

Myelin management by the 18.5‐kDa and 21.5‐kDa classic myelin basic protein isoforms

The classic myelin basic protein (MBP) splice isoforms range in nominal molecular mass from 14 to 21.5 kDa, and arise from the gene in the oligodendrocyte lineage (Golli) in maturing oligodendrocytes. The 18.5‐kDa isoform that predominates in adult myelin adheres the cytosolic surfaces of oligodendrocyte membranes together, and forms a two‐dimensional molecular sieve restricting protein diffusion into compact myelin. However, this protein has additional roles including cytoskeletal assembly and membrane extension, binding to SH3‐domains, participation in Fyn‐mediated signaling pathways, sequestration of phosphoinositides, and maintenance of calcium homeostasis. Of the diverse post‐translational modifications of this isoform, phosphorylation is the most dynamic, and modulates 18.5‐kDa MBP's protein‐membrane and protein‐protein interactions, indicative of a rich repertoire of functions. In developing and mature myelin, phosphorylation can result in microdomain or even nuclear targeting of the protein, supporting the conclusion that 18.5‐kDa MBP has significant roles beyond membrane adhesion. The full‐length, early‐developmental 21.5‐kDa splice isoform is predominantly karyophilic due to a non‐traditional P‐Y nuclear localization signal, with effects such as promotion of oligodendrocyte proliferation. We discuss in vitro and recent in vivo evidence for multifunctionality of these classic basic proteins of myelin, and argue for a systematic evaluation of the temporal and spatial distributions of these protein isoforms, and their modified variants, during oligodendrocyte differentiation.