bluuebunny/crossref_metadata_2025 huggingface dataset:
Insulin/insulin‐like growth factor I and other epigenetic modulators of myelin basic protein expression in isolated oligodendrocyte progenitor cells
Authors: Saneto R. P. , Low K. G. , Melner M. H. , de Vellis J.
Published: January 2005

The expression of myelin basic protein by the oligodendrocyte is an integral event in the maturation of central nervous sytem function. Although much is known concerning the various myelin basic protein species, their temporal expression, and processing of RNA transcripts, little is known about the epigenetic factors responsible for the regulation of myelin basic protein (MBP) expression. In this study, we present evidence that insulin/insulin‐like growth factor‐I can increase the levels of MBP protein in isolated oligodendrocyte progenitor cells cultured in a serumless, chemically defined medium (ODM). Insulin was found to increase MBP protein in a dose‐responsive manner, reaching a maximal level at 72 hr of exposure. Both insulin‐like growth factor‐I (IGF‐I) and insulin were demonstrated to have no effect on MBP RNA levels. These data indicate that insulin/IGF‐I increased MBP protein levels at a level distal to transcription The dose response of insulin action suggests that it may have a MBP regulatory function, distinct from IGF‐I. When added individually, the other supplements of ODM, transferrin (500 ng/ml), and basic fibroblast growth factor (5 ng/ml) had no effect on MBP expression. However, when all three components were combined, a synergistic effect resulting in increased MBP protein and total RNA levels was found. The phorbol ester 12‐O‐tetradecanoyl phorbol acetate was found to reduce intracellular MBP RNA levels. The cAMP analogue/dibutyryl cAMP had contrasting effects on MBP RNA levels; no effect occurred in cultures grown in fetal calf serum, but a reduction in RNA levels was found in cultures grown in ODM. These data suggest that only a select range of extrinsic factors may be involved in MBP regulation, and depending on the environmental milieu, epigenetic agents may modulate gene activity differently.
Differential expression of galactocerebroside, myelin basic protein, and 2′,3′‐cyclic nucleotide 3′‐phosphohydrolase during development of oligodendrocytes in vitro
Authors: Knapp P. E. , Skoff R. P. , Sprinkle T. J.
Published: January 2005

This paper describes the differential expression and localization of myelin components within membrane sheets produced by oligodendrocytes in vitro. In double‐labeling experiments using antibodies to the myelin antigens 2′,3′‐cyclic nucleotide 3′‐phosphohydrolase (CNP) and galactocerebroside (GC), the two antigens were coexpressed in at least 95% of oligodendrocytes at all ages examined. A small population of relatively undifferentiated cells expressed one antigen before the other. Within the membrane sheets produced by the cultured cells, CNP and GC are distributed differently. CNP is highly concentrated in cell bodies, in a network of processes extending from the cell body into the sheets, and around the perimeter of the sheets. CNP staining cannot be detected in some areas within the body of the sheet. When present, it is of low intensity. Under our labeling conditions, GC staining is found throughout the membrane sheets, except in the network of veins which are CNP+. GC and myelin basic protein (MBP) staining are seen in similar membrane domains even though GC is a surface component while MBP resides on the cytoplasmic face.

Both the timing and localization of CNP immunostaining show that CNP is as early a marker for oligodendrocytes as GC, and support the idea that CNP may play a structural role in the myelin membrane. Double‐labeling studies with GC and CNP antibodies also show that the true shape of a cell and the extent of its development are not always revealed by a single antigen. The differential distribution of antigens within membrane sheets illustrates that they contain areas of structural specialization that may reflect the situation in intact myelin.
Peripheral motoneuron interactions with laminin and schwann cell‐derived neurite‐promoting molecules: Developmental regulation of laminin receptor function
Authors: Tomaselli K. J. , Reichardt L. F.
Published: January 2005

Schwann cells synthesize several neurite outgrowthpromoting molecules and localize them in either the extracellular matrix (ECM; e.g., laminin) or on the plasma membrane (e.g., L1/NgCAM and N‐cadherin). Neurite outgrowth by embryonic chick ciliary ganglion (CG) neurons in response to these Schwann cell molecules largely depends on several specific neuronal cell surface receptors: integrin β1‐class ECM receptors, L1/NgCAM, and N‐cadherin (Bixby et al.: Journal of Cell Biology 107:353–361 1988). To address whether neuronal ECM receptors are regulated independently of cell surface adhesion molecules, we studied the ability of dissociated CG neurons from different developmental ages to extend neurites rapidly on (1) substrates coated with the ECM glycoprotein laminin (either from Schwann cell‐conditioned medium or purified from the Engelbreth‐Holm Swarm sarcoma) or (2) the surfaces of Schwann cells or Schwannoma (RN22) cells. CG neurons gradually lost the ability between embryonic day 8 (E8) and E14 to attach to and extend neurites in an integrin‐dependent fashion on purified laminin or Schwann cellderived laminin. The inability of E14 CG neurons to respond to laminin was partially reversed after explantation for 2.5 days in vitro, which increased the percentage of responsive neurons approximately tenfold. E14 neurons remained capable of extending neurites rapidly on the surfaces of Schwann and Schwannoma cells. Thus, the inability of E14 neurons to respond to laminin reflects a specific loss of laminin receptor function, while other receptors, most likely N‐cadherin and L1/NgCAM, remain capable of promoting neurite outgrowth on Schwann cell surfaces. Since integrin β1‐class heterodimers have been shown to function directly as receptors mediating neuronal attachment and process outgrowth on laminin, our results imply that the expression or function of laminin‐binding integrin heterodimers is regulated during the development of CG neurons. The apparent loss of integrin receptor function occurs during the period when the axons of CG neurons innervate their targets. Substantial integrin receptor function is recovered when target contact is disrupted by explantation. Thus, the functions of integrin‐class receptors in CG neurons may be regulated by target contact.
A gradient molecule in developing rat retina: Expression of 9‐O‐acetyl GD3 in relation to cell type, developmental age, and GD3 ganglioside
Authors: Sparrow J. R. , Barnstable C. J.
Published: January 2005

In the embryonic and postnatal rat retina a cell surface antigen that is detected by monoclonal antibody JONES is distributed in a dorsoventral gradient. Biochemical analysis has determined that the antigen is a modified ganglioside, 9‐O‐acetyl GD3. In the present study, the distributions of 9‐O‐acetyl GD3 and its possible precursor GD3 in developing rat retina were compared immunocytochemically using specific monoclonal antibodies JONES and R24. On embryonic day 13 (E13) immunoreactivity to JONES was localized to central retina; however, R24 stained throughout the retinal epithelium. By E20, when JONES binding was distributed in a gradient along the dorsoventral axis, R24 again stained dorsal and ventral regions with uniform intensity. Analysis of freshly dissociated retinal cells further revealed that GD3 and 9–O‐acetyl GD3 expressions do not necessarily coincide. At E15 and postnatal day 2 (PN2), the majority of cells (78 and 92%, respectively) were immunolabeled by antibody to GD3, while between E15 and PN2 the percentage of cells immunolabeled by antibody to 9–O‐acetyl GD3 rose from 19 to 68%. By PN4, labeling decreased for both molecules; however, the rate of decline in 9–O‐acetyl GD3 labeling was more pronounced. Regulation of the numbers of JONES‐positive cells does not appear to depend on interaction with the extraretinal environment, for in neural retina explanted at E15 the proportions of 9–O‐acetyl GD3–bearing cells was found to be similar to the percentage observed in neural retina developing to an equivalent age in vivo. Experiments to identify the retinal cell types bearing 9‐O‐acetyl GD3 revealed that it is expressed by both neurons and glia growing in monolayer cultures of rat perinatal neural retina. Examination of freshly dissociated retinal cells following simultaneous labeling for some specific cell types and 9–O‐acetyl GD3 demonstrated that the latter determinant is present on photoreceptor, amacrine, and ganglion cells. For each neuronal cell type, however, not all of the cells were immunoreactive with JONES.

We conclude that the differences in the percentages of JONES‐ and R24‐positive cells, and in particular the different rates at which JONES and R24 staining declined with age, indicate that the expression of the JONES epitope is regulated with some independence from parent ganglioside GD3. In addition, since JONES is expressed on a variety of retinal cells, both neurons and glia, and since the rate of decline in proportions of JONES‐labeled cells was found to be the same for at least two neuronal cell types, regulation of the expression of the JONES epitope, in keeping with its dorsoventral gradient of distribution, is probably not based on cell type.
Regulation of Schwann cell nerve growth factor receptor by cyclic adenosine 3′,5′‐monophosphate
Authors: Mokuno K. , Sobue G. , Reddy U. R. , Wurzer J. , Kreider B. , Hotta H. , Baron P. , Ross A. H. , Pleasure D.
Published: January 2005

Previous studies indicated that Schwann cells in immature nerves express nerve growth factor (NGF) receptors, and that this expression is down regulated during development but re‐induced by Wallerian degeneration. It was also shown that immature Schwann cells are induced to express galactocerebroside and other molecules characteristic of mature Schwann cells by either contact with an axon or treatment with the cyclic adenosine 3′,5′‐monophosphate (cAMP) analogues dibutyryl cAMP (dbcAMP) and 8‐bromo cAMP or the adenylate cyclase activator forskolin. In the present study, NGF receptors on the surface of cultured Schwann cells were demonstrated by binding of an anti‐rat NGF receptor monoclonal antibody or of radioiodinated NGF. Treatment of cultured Schwann cells with cAMP analogues or forskolin resulted in a progressive decrease in both immunoreactive NGF receptors and radioiodinated NGF binding. The cultured Schwann cells contained a polyadenylated RNA species homologous with human melanoma NGF receptor mRNA in sequence and size. The amount of this NGF mRNA was lower in cAMP analogue‐treated than in untreated Schwann cells.
Role of protein kinase C in glial cell proliferation
Authors: Bhat N. R.
Published: January 2005

Phorbol 12‐myristate 13‐acetate (PMA) has been shown to stimulate DNA synthesis and cell proliferation in a population of glial cells isolated from newborn rat brain. The non‐tumor promoter 4α‐phorbol 12,13‐didecanoate (4α‐PDD), on the other hand, was without an effect. The cultures treated with PMA displayed an extensive process formation and an increase in cell content. The tumor promoter‐induced [3H]thymidine incorporation into acid‐precipitable material was completely blocked by 1‐(5‐isoquinolinylsulfonyl)‐2‐methylpiperazine (H‐7), a potent inhibitor of protein kinase C (PKC), thereby suggesting a role for PKC in the control of DNA synthesis in glial cells. Subcellular fractionation and in vitro assay of PKC activity revealed a translocation of the enzyme from cytosol to particulate fraction in PMA‐treated cultures.
Neurotoxic effect induced by quinolinic acid in dissociated cell culture of mouse hippocampus
Authors: Khaspekov L. , Kida E. , Victorov I. , Mossakowski M. J.
Published: January 2005

Quinolinic acid (QUIN), an endogenous convulsant that is a product of tryptophan metabolism, is suggested to belong to the pathogenic factors in some neurodegenerative disorders of the central nervous system (CNS). The aim of the present study was to evaluate the effect of QUIN on hippocampal nerve cells in dissociated cell culture at different periods of its development in vitro. The neurodegenerative effect of QUIN in vitro was found 1.5 to 2 hr after exposure to QUIN (500 μM) in differentiated hippocampal culture 20–21 days in vitro (DIV). The cytotoxic action depended on the nature of the interneuronal relations established in culture and on the period of neuronal development in vitro. Neurons in 11‐DIV cultures were undamaged even 10 days after exposure to QUIN; nor were the individual, isolated neurons probably not connected synaptically with the other ones in 20–21 DIV cultures. Ultrastructural analysis of 20–21 DIV cultures exposed to QUIN showed acute swelling and destruction of postsynaptic elements and severe degeneration of individual nerve cells located in cellular aggregates, whereas presynaptic axon terminals remained intact. The results emphasize the utility of QUIN as a tool to model degenerative changes of CNS and confirm the participation of mature synaptic junctions in QUIN neurotoxicity.
Compartmentalization of acetylcholinesterase in the chick retina
Authors: Ramírez G. , Barat A. , Donoso J. A. , Fernández H. L.
Published: January 2005

Using selective inhibitor treatments, we have studied the distribution of asymmetric (A) and globular (G) forms of acetylcholinesterase (AChE) in the extra‐ and intracellular compartments of chick retina, a specialized region of chick central nervous system (CNS). Our results show that the chick retinal collagen‐tailed AChE (an example of class II asymmetric molecular forms) is essentially an extracellular form of the enzyme; this is the first demonstration of the extracellular localization of asymmetric AChE in the vertebrate CNS. The active site of most of the hydrophobic, membrane‐bound G4‐form is also exposed to the external environment. In turn, the smaller molecular weight G‐forms (G2 and G1) are localized within the cells, where they may represent intermediate components in the assembly or degradation of the more complex enzymatic molecular species.

Histoenzymatic ultrastructural techniques show internal AChE in amacrine as well as in ganglion cell bodies, and external enzyme, specifically associated with synapses and axons, in the inner plexiform layer. The probable cooperation of the extracellular A12‐forms and the membrane‐bound G species (mainly G4) of the enzyme to the hydrolysis of acetylcholine (ACh) released into the external compartment is suggested and discussed.
A volume‐dependent, chloride—sensitive component of taurine release stimulated by potassium from retina
Authors: Domínguez L. , Montenegro J. , Pasantes‐Morales H.
Published: January 2005

Chick retinas exposed to 56 mM KCl showed marked swelling of cells and synaptic structures, detected by morphological examination as well as by measurements of cell water content. Retinal cell swelling is prevented by omission of chloride. Potassium chloride, 56 mM, elicited the efflux of [3H]taurine by a process that is partly calcium‐dependent, highly chloride‐dependent (80%), and sensitive to furosemide. The release of [3H]dopamine under the same conditions is completely calcium‐dependent and increases in the absence of chloride. The chloride‐dependent component of [3H]taurine efflux seems related to increases in volume changes since it is markedly reduced when cell swelling is prevented either by maintaining constant the K × Cl product (Donnan equilibrium) or by making the solution hypertonic with mannitol. Omission of calcium and of chloride showed additive effects, practically suppressing the potassium‐stimulated release of taurine. These results suggest that the potassium‐stimulated efflux of [3H]taurine consists of a large component associated to changes in cell volume and a small component probably related to the depolarizing effects of potassium.
Chicken NGF and Non‐NGF trophic factor synthesis and release by sciatic nerves in vitro
Authors: Ferguson I. A. , Williams R. , Rush R. A.
Published: January 2005

The time course of production and release of nerve growth factor (NGF) and non‐NGF neuronotrophic factors for sympathetic neurons by chicken and rat sciatic nerves in culture was examined. These tissues actively synthesize and release neuronotrophic activity as metabolically poisoning nerves with azide dramatically reduced the amount of trophic activity released into the culture medium. The sustained release of this activity also was shown to be dependent on the presence of low‐molecular‐weight dialysable molecules present in foetal calf serum and amniotic fluid from day 11 chicken embryos. Affinity‐purified antimouse NGF antibodies were used to show that sciatic nerves in culture release both NGF and non‐NGF trophic factors. These antibodies inhibited all bioactivity of both mouse NGF and of a partially purified preparation of chicken NGF. Immunoblot studies confirm that the antibodies recognize both rodent and avian NGF. Excess antibody inhibited only about 50% of the trophic activity in media conditioned over rat or chicken nerves for the first 24 hr. Relatively similar amounts of this non‐NGF trophic activity were released throughout 6 days in culture, and this trophic activity kept sympathetic neurons alive in culture in the absence of NGF for more than 4 days. NGF levels were quantified with a two‐site enzyme‐linked immunoassay and found to parallel changes in NGF bioactivity. Rat nerves released increasing amounts of NGF with time in culture. Whole chicken sciatic nerves, however, released decreasing amounts of NGF with time in culture, but when these nerves were desheathed by removal of the epineurium and attached tissue, the pattern of NGF release was similar to that observed in the smaller rat sciatic nerves. These studies therefore characterize antibodies recognising chicken NGF, demonstrate that peripheral nerve tissue synthesize trophic factors other than NGF, and identify factors that influence NGF synthesis.
Effect of nerve growth factor on the synthesis of amino acids in PC12 cells
Authors: Zielke H. R. , Tildon J. T. , Kauffman F. C. , Baab P. J.
Published: January 2005

Radioactive short‐chain fatty acids preferentially label glutamine relative to glutamate in brain due to compartmentation of glutamine and glutamate. To determine whether this phenomenon occurs in a single cell culture model, we examined the effect of fatty acid chain length on the synthesis as well as pool size of selected amino acids in rat pheochromocytoma PC12 cells, a cell culture model of the large glutamate compartment in neurons. Intracellular 14C‐amino acids were quantitated by HPLC, and the incorporation of [U‐14C]‐glucose, [1minus;14C]‐butyrate, [1‐14C]‐octanoate, and [1‐14C]‐palmitate into five amino acids was measured in native and NGF‐treated PC12 cells. NGF pretreatment decreased the intracellular concentration of amino acids as did addition of fatty acids but these effects were not additive. Specific activities of amino acids in native cells labelled by 14C‐octanoate were 1,300 DPM/nmol, 490 DPM/nmol, 200 DPM/nmol, and 110 DPM/nmol for glutamate, aspartate, glutamine, and serine, respectively. No radioactivity was detected in alanine. Similar specific activities were noted when 14C‐butyrate was the precursor; however, there was at least 5‐fold less if 14C‐palmitate was the precursor. Pretreatment of cells with NGF decreased the specific activity of amino acids by 25–65%. Specific activities of amino acids synthesized from 14C‐glucose decreased in the following order: glutamate, 1,640 DPM/nmol; aspartate, 1,210 DPM/nmol; alanine, 580 DPM/nmol; glutamine, 275 DPM/nmol; and serine, 80 DPM/nmol for native cells. NGF pretreatment decreased the specific activities of glutamate and glutamine, but not of the other 3 amino acids. The preferred precursor for glutamate synthesis in native PC12 cells was glucose followed by octanoate, butyrate and palmitate (16:6:3:1).
Nerve regulation of class I and Class II‐asymmetric forms of acetylcholinesterase in rat skeletal muscles
Authors: Fadić R. , Inestrosa N. C.
Published: January 2005

Two classes of collagen‐tailed, asymmetric forms (A‐forms) of acetylcholinesterase (AChE) have been described in skeletal muscles of vertebrates. They are distinguished by their different solubilization requirements: class I A‐forms are solubilized in the presence of high salt, whereas class II A‐forms require in addition a chelating agent for solubilization. We report here that class II A‐forms are less sensitive to nerve section than are class I A‐forms. The latter form decreases faster and to a lower level of activity after denervation. The decay of both AChE classes is more rapidly in short‐stump nerves than in long ones. The effect of nerve section on class II A‐forms appears to be dependent on the particular muscle group being studied. Both classes of A‐forms reappear after muscle reinnervation, but the class I A‐forms recovered earlier. More interestingly, both classes of A‐forms increase in normally innervated skeletal muscles after contralateral nerve injury. In this case, however, the class II A‐forms change first. Muscular disuse induced by contralateral tenotomy is also followed by a rise in class II A‐forms. Our results, showing differences in response and flexibility in the changes of the two classes of A‐forms in several experimental conditions, represent a relevant contribution to the understanding of the regulation and functional role of the asymmetric forms of AChE in vertebrate skeletal muscles.
Induction of protooncogene fos by extracellular signals in primary glial cell cultures
Authors: Condorelli D. F. , Kaczmarek L. , Nicoletti F. , Arcidiacono A. , Dell'Albani P. , Ingrao F. , Magrì G. , Malaguarnera L. , Avola R. , Messina A. , Stella A. M. Giuffrida
Published: January 2005

In the present study various extracellular factors, acting through different second messenger systems, were examined for their capacity to increase the level of c‐fos mRNA in primary glial cell cultures. In particular EGF, 12‐O‐tetradecanoylphorbol 13‐acetate, the beta‐adrenergic agonist isoproterenol, and the glutamate agonists, ibotenic and quisqualic acid, were studied. All the extracellular stimuli tested induced a rapid and transient increase in c‐fos mRNA level in glial cell cultures regardless of the signal transduction pathway and the final effect on cell proliferation.
Immunohistochemistry with anti‐calbindin and anti‐neurofilament antibodies in the cerebellum of methylazoxymethanol‐treated mice
Authors: de Barry J. , Gombos G.
Published: January 2005

Mice pups were injected with methylazoxymethanol at birth (MAM0) or on the fifth postnatal day (MAM5) and their cerebella were examined when adult. Immunohistochemistry with an antiserum directed against calbindin, a protein specific for Purkinje cells, was used to survey more easily Purkinje cell position and orientation. For a general view of basket cell axon distribution, we used a monoclonal antibody that recognized the phosphorylated form of the 200 kD constituent protein of neurofilaments, which is axon specific. The present results confirm that in MAM5 the cytoarchitecture was preserved, some Purkinje cells degenerated, and the pericellular basket around the Purkinje cells was apparently normal. In MAM0 animals, the Purkinje cells appeared malpositioned and disoriented, the pinceau around the Purkinje cell hillock was absent, but basket cell axons were present. This indicated that the absence of pinceau was not due to the absence of basket cells, but probably to alterations of cell interactions, which hindered the proper pericellular basket formation.
Human brain microvascular DR‐antigen
Authors: Pardridge W. M. , Yang J. , Buciak J. , Tourtellotte W. W.
Published: January 2005

Antigen presentation within the human central nervous system by the class II histocompatibility DR‐antigen may take place at either the brain capillary–endothelial interface or at perivascular cells, such as smooth muscle or pericytes. The present studies employ a new sensitive immunoperoxidase technique, a mouse monoclonal antibody to the human DR‐antigen, and microvessels isolated from autopsied human brain. The experiments show that the DR‐antigen is readily detectable in human microvasculature of normal brain and is found in the smooth muscle pre‐capillary arterioles and capillary pericytes with minimal, if any, staining of capillary endothelium. These results are consistent with the hypothesis that antigen presentation in the CNS occurs primarily at a site immediately distal to the blood–endothelial interface.
Indole amine deficiency in blood and cerebrospinal fluid from patients with human immunodeficiency virus infection
Authors: Larsson M. , Hagberg L. , Norkrans G. , Forsman A.
Published: January 2005

Twenty‐four patients with human immunodeficiency virus (HIV) infection were investigated for possible changes in certain indole amine constituents in blood and cerebrospinal fluid (CSF). Albumin in serum was determined and used as a rough nutritional marker. Six of the 24 patients had acquired immunodeficiency syndrome AIDS, four had other clinical symptoms of HIV infection, and 14 had no apparent symptoms. The HIV‐seropositive patients had significantly decreased tryptophan values; their blood concentrations were 28% lower and their CSF concentrations 30% lower than corresponding values in 14 healthy controls. The blood concentrations of 5‐hydroxytryptamine (5‐HT) were 50% lower, and the platelet content of 5‐HT was 36% lower in HIV‐infected individuals than in the control group. The most pronounced changes were invariably seen in the six cases with AIDS and in patients with a low number of CD4+ cells. No significant difference between controls and HIV‐seropositive patients was detected in the mean CSF concentrations of 5‐hydroxyindoleacetic acid (5‐HIAA), although these levels were markedly reduced in four of the HIV patients. Neither was any significant difference seen between patients and controls in the serum concentrations of albumin.
Functional expression of the yes proto‐oncogene protein in Xenopus oocytes injected with chicken cerebellar mRNA
Authors: Sudol M.
Published: January 2005

Using monospecific antibodies raised against the amino terminal domain of viral‐yes protein, we precipitated a 58 kD protein‐tyrosine kinase from the brain of Xenopus laevis frog. By a number of criteria, including pattern of expression in various tissues and one‐dimensional peptide mapping, we concluded that pp58 is very likely the authentic amphibian yes protein because it is more similar to the chicken yes protein than to any other known tyrosine kinases. The pp58c–yes is expressed in adult brain at elevated levels. In contrast, its level of expression in follicular and denuded oocytes is 30–50 times lower. Because of the low endogenous expression of the oocyte‐associated yes kinase, we were able to transiently express and analyze pp62c‐yes by injecting oocytes with 27S size fraction of poly(A)+ mRNA isolated from chicken cerebella. The pp62c‐yes expressed in oocytes from the exogenous message comigrated on SDS polyacrylamide gel with pp62c‐yes from cerebellum and was indistinguishable from it by one‐dimensional peptide mapping. The pp62c‐yes expressed in oocytes was enzymatically active. A number of phosphotyrosine‐containing proteins were detected specifically in the c‐yes mRNA‐injected oocytes. The usefulness of the Xenopus oocyte expression system to study the functional aspects of c‐yes protein and other tyrosine kinases was evaluated.
X‐ray diffraction analysis of myelin lipid/proteolipid protein multilayers
Authors: Brown F. R. , Karthigasan J. , Singh I. , Kirschner D. A.
Published: January 2005

To examine the proposal that myelin proteolipid protein underlies the adhesion of neighboring membranes in central nervous system (CNS) myelin, we carried out X‐ray diffraction studies on the structure and interactions of model bilayers composed of total myelin lipids plus proteolipid apoprotein (PLP). Total myelin lipids were used because their heterogeneity was expected to provide an appropriate environment for the integral membrane protein to achieve its native conformation and establish appropriate contacts with the apposed bilayer.

We found that incorporation of PLP into myelin lipid bilayers, whether organized into multilamellar vesicles or oriented multilayers, did not appreciably affect the lamellar period, which ranged from 65–71 Å. In oriented multilayers, the wide‐angle spacing at ≈4.8 Å, which arises from the lateral packing of lipid chains and is perpendicular to the lamellar diffraction, was less oriented and weaker in bilayers containing the protein. These results indicate that PLP was incorporated into the bilayers and had a disordering effect on the hydrocarbon chains but did not extend into the spaces between bilayers. Bilayer profiles calculated from the lamellar diffraction to about 15 Å spacing did not show any major changes in the distribution of electron density, suggesting that to moderate resolution, the protein was distributed uniformly across the width of the lipid bilayer. Periodicities measured from osmotically stressed multilamellar vesicles did not depend on the presence of PLP, indicating that the protein did not form stabilizing contacts between bilayers. We suggest that the results of structural, biophysical, and functional studies on model bilayers containing proteolipid protein may not extrapolate to the in vivo interactions of myelin because it is unlikely that the protein is in its native conformation.
Defective T helper cell epitope responsible for the failure of region 69–84 of the human myelin basic protein to induce experimental allergic encephalomyelitis in the lewis rat
Authors: Hashim G. A. , Galang A. B. , Srinivasan J. V. , Carvalho E. F. , Offner H. , Vandenbark A. A. , Cleveland W. L. , Day E. D.
Published: January 2005

Studies from our laboratory have shown that region 69–84 (synthetic peptide S49S) of myelin basic protein (MBP) defines an encephalitogenic sequence for experimental allergic encephalomyelitis (EAE) in Lewis rats. The most potent EAE inducers are the guinea pig MBP (Gp‐MBP) and region 69–84, known as synthetic peptide Gp‐S49S:
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Human (H‐MBP) was considerably less potent than Gp‐MBP, and region 69–84 (H‐S49S) of H‐MBP did not induce hind leg paralysis or any histological signs of EAE. Since the development of EAE requires the expression of specific T and B cell epitopes, sequence analysis of H‐S49S and Gp‐S49S revealed phylogenetic variations in the H‐S49S sequence, characterized by deletion of Gln (No.76), insertion of His‐Gly at positions 77 and 78, and substitution of Ser with Thr at position 80:
equation image
Like Gp‐S49S, peptide H‐S49S induced the formation of antibodies with specificities directed against the C‐terminal of the H‐S49S, Gp‐S49S, and homologous sequences. In contrast to Gp‐S49S, neither H‐S49S nor shorter peptides induced clonal T cell expansion when either of the peptides was added to encephalitogenic T cell clone D in culture. Clone D, which expresses T helper phenotype, was selected from encephalitogenic peptide‐primed Lewis rats. The results of the study show that the failure of H‐S49S to induce EAE is related to sequence alterations in the T helper cell epitope but not in the B cell epitope located in the N‐ and C‐terminal portions of the S49S sequence, respectively. Also, the results suggest that the EAE activity of H‐MBP may be related to either a second EAE region that expresses T helper and B cell functions or to a dislocated T helper cell epitope that operates in conjunction with the B cell epitope located in the C‐terminal region of the H‐S49S sequence.
Ethanol alters the transfer of arachidonic acid to ethanolamine plasmalogens in C‐6 glioma cells
Authors: Sun G. Y. , Aradottir S. , Gustavsson L. , Alling C.
Published: January 2005

In this study, the effects of ethanol exposure on uptake and metabolism of arachidonic acid by C‐6 glioma cells in culture was examined. Labeled arachidonic acid was effectively taken up by the phospholipids of these cells and radioactivity was initially incorporated into phosphatidylinositols and phosphatidylcholines, reaching a peak between 4 and 6 hours. However, the labeling of ethanolamine plasmalogens continued to show an increase with time after labeled arachidonic acid has been exhausted in the medium. Since over 90% of labeled arachidonic acid was already taken up by the cells after 4 hours of exposure, the continued increase in labeling of ethanolamine plasmalogens is attributed to a transacylation mechanism. Cells grown in 150 mM ethanol for 2 days did not show a change in the overall incorporation of labeled arachidonic acid into phospholipids but showed a significant increase in labeling of ethanolamine plasmalogens, which was marked by a concomitant decrease in labeling of phosphatidylcholines. Ethanol exposure also resulted in a significant increase in the transfer of labeled arachidonic acid to triacylglycerols. Changes in phospholipid and triacylglycerol labeling pattern positively correlated with increasing ethanol concentration from 75 to 300 mM. Besides, most ethanol effects were readily noticeable after 24 hours of exposure. These data suggest a specific effect of ethanol on promoting the transacylase process for biosynthesis of ethanolamine plasmalogens as well as the acyltransferase for biosynthesis of triacylglycerols.
Effect of repeated electroconvulsive treatment on regional concentrations of tachykinins, neurotensin, vasoactive intestinal polypeptide, neuropeptide Y, and galanin in rat brain
Authors: Stenfors C. , Theodorsson E. , Mathé A. A.
Published: January 2005

The effects of single and repeated electroconvulsive treatments (ECTs) on brain regional distribution of substance P (SP), neurokinin A (NKA), neurotensin (NT), neuropeptide Y (NPY), vasoactive intestinal polypeptide (VIP), and galanin (GAL) were studied in the rat. Rats were divided into four groups receiving one of the following treatments: one ECT, one sham ECT, six ECTs, or six sham ECTs. After sacrifice by focused microwave irradiation, brains were dissected into frontal cortex, striatum, occipital cortex, hippocampus, hypothalamus, and pituitary sections. Peptides were extracted by boiling the tissues in 1 mol/l acetic acid and measured in extract aliquots by specific radioimmunoassays. Marked regional differences (P =.0005) were found for each of the peptides measured. The highest concentrations of SP and NKA were found in the hypothalamus and, in descending order, in striatum, pituitary, frontal cortex, occipital cortex, and hippocampus. For NT, the highest level was found in the hypothalamus and, in descending order, in pituitary, striatum, hippocampus, frontal cortex, and occipital cortex. The highest VIP concentrations were measured in frontal and occipital cortex, followed by pituitary, hypothalamus, striatum, and hippocampus. The highest NPY and GAL concentrations were found in the hypothalamus and the pituitary; in frontal and occipital cortex, as well as in the striatum and hippocampus, the peptides levels were rather evenly distributed. Repeated handling (stress?) decreased both NT and GAL in frontal (P =.05 and.04) and occipital cortex (P =.09 and.07). ECT, one or six treatments, had no effect on SP, NKA, NT, VIP, or GAL concentrations in different brain regions. However, an increase in NPY concentrations in hippocampus (six sham ECTs vs. six ECTs = right: 112 ± 61 vs. 246 ± 159, and left: 143 ± 42 vs. 303 ± 160 pmol/g; P =.0005) and occipital cortex (right: 118 ± 42 vs. 167 ± 98; and left: 97 ± 45 vs. 157 ± 42 pmol/g, P =.003) was obtained after six ECTs but not following a single treatment. The results demonstrate a regional effect of ECT on NPY and raise the possibility that one of the ECTs' modes of action may be by elevating NPY levels in the CNS.
Sequence of a human MAP‐2 region sharing epitopes with Alzheimer neurofibrillary tangles
Authors: Dammerman M. , Yen S.‐H. , Shafit‐Zagardo B.
Published: January 2005

Microtubule‐associated protein 2 (MAP‐2), an abundant neuronal protein, consists of a short microtubule‐binding domain and a long projection arm. MAP‐2 shares epitopes with Alzheimer neurofibrillary tangles (NFT). However, most anti‐MAP‐2 antibodies do not stain detergent‐extracted NFT, and the role of MAP‐2 in NFT formation has therefore been unclear. We have determined the sequence of a 1.7 kb partial MAP‐2 cDNA encoding at least three NFT epitopes. The epitopes are not removed by detergent extraction of tangle preparations, suggesting that they are integral components of NFT. Expression vectors containing restriction fragments of the cDNA were used to assign the epitopes to a 51‐amino‐acid region near the end of the MAP‐2 projection arm, distal to the microtubule.
Neurite‐promoting influences of proliferating schwann cells and target‐tissues are not prerequisite for rapid axonal elongation after nerve crush
Authors: Bresjanac M. , Sketelj J.
Published: January 2005

An important role in peripheral nerve regeneration has been ascribed to humoral trophic and tropic agents arising from the nonneuronal cells in the distal nerve stump and the denervated targets. In order to estimate their contribution to axonal elongation after crush injury to the rat sciatic nerve, an in vivo model was designed in which local cellular and target‐derived influences were eliminated by (1) freeze‐thawing of a long nerve segment distal to the crush site and (2) cutting the nerve far distally to the crush site, but within the frozen‐thawed segment, and deflecting the frozen‐thawed nerve stump in the opposite direction from its natural course.

The sensory and motor axon elongation rate was estimated from the results of the nerve pinch test and choline acetyltransferase distribution along the nerve segment distal to the crush.

The elongation rate of regenerating axons in deflected nerve segments, either non‐treated or frozenthawed, was close in magnitude to that obtained when target‐derived influences were not eliminated. Neurotropism of axonal targets is therefore of little importance for axon elongation after nerve crush. In the absence of Schwann cells along the axonal path in frozen‐thawed nerve segments, the elongation rate of both sensory and motor axons declined by about 40%. This implies that interactions between viable Schwann cells and growth cones of regenerating axons are not prerequisite for rapid axon elongation when Schwann cell basal lamina constitutes the growth substratum. Nevertheless, Schwann cells in Bungner bands possibly enhance the axon elongation rate by humoral or cell surface‐mediated mechanisms.
Phorbol ester‐induced change in astrocyte morphology: Correlation with protein kinase C activation and protein phosphorylation
Authors: Harrison B. C. , Mobley P. L.
Published: January 2005

Treatment with 300 nM phorbol 12‐myristate 13‐acetate (PMA) transforms polygonal‐shaped cultured astrocytes into process‐bearing cells and produces a shift in protein kinase C (PK‐C) from the cytosol to the membrane. Exposure to PMA also produces increases in the phosphorylation of several proteins including vimentin, glial fibrillary acidic protein (GFAP), an acidic 80,000 molecular weight protein, and two 30,000 molecular weight proteins (pI5.5 and 5.7). The effects of PMA on the translocation of PK‐C and on protein phosphorylation precede the PMA‐induced changes in astrocyte morphology, and a close correlation exists between the concentration of PMA necessary to elicit half‐maximal and maximal effects on the shift of PK‐C to the membrane and on protein phosphorylation. In addition, the PMA‐induced alterations in cell morphology are not permanent, and within 24 hr after PMA treatment the cells have reverted almost to their original morphology. A second exposure to PMA at this time fails to elicit further change in cell shape and is also incapable of producing increases in the phosphorylation of proteins. It was determined that there is little, if any, PK‐C present in these PMA‐pretreated cells. The morphological responsiveness to PMA gradually returns in 5 to 8 days after the initial treatment with PMA, and this is accompanied by the recovery of PK‐C activity and the phosphorylation response. Therefore, these studies suggest that the effect of PMA on astrocyte morphology is mediated by the activation of PK‐C and subsequent protein phosphorylation.
Interspecies differences in the substance P‐ and vasoactive intestinal polypeptide‐like immunoreactivities in the olfactory bulb ofSalmo gairdneri and Barbus meridionalis
Authors: Alonso J. R. , Covenas R. , Lara J. , De Leon M. , Arevalo R. , Aijon J.
Published: January 2005

The distribution of substance P‐like (SP) and vasoactive intestinal polypeptide‐like (VIP) structures was studied using an indirect immunoperoxidase technique in the mediterranean barbel Barbus meridionalis and in the rainbow trout Salmo gairdneri. SP‐like positive fibers were observed in the inner strata of the mediterranean barbel olfactory bulb, mainly the granule cell and the plexiform layers. Ganglion cells and fibers of the terminal nerve were also labeled. No SP‐positive structure was found in the olfactory bulb of the rainbow trout. On the contrary, the VIP antiserum used displayed very strong immunostaining in the olfactory nerve fiber layer of Salmo gairdneri, whereas those fibers in Barbus meridionalis showed no immunoreactivity. After a complete transection of the olfactory tract and a survival time of 20 days, SP‐immunostained fibers were not observed. Thus, they can presumably be identified as centrifugal fibers, coursing from the telencephalic hemispheres through the olfactory tracts, into the olfactory bulb. The VIP immunoreactivity was confined to the olfactory fibers, both in the olfactory nerve and the olfactory bulb. The positive immunostaining disappeared after chemical lesion of the olfactory mucosa. These observations demonstrate that the olfactory bulb of freshwater teleosts exhibits a high degree of heterogeneity in its immunocytochemical distribution pattern, this pattern also differing from previous reports on higher vertebrates.
Asymmetry of interhemispheric responses evoked in the prefrontal cortex of the rat
Authors: Pérez H. , Ruiz S. , Ernández A. H. , Soto‐Moyano R.
Published: January 2005

Interhemispheric responses of anesthetized rats were evoked in the prefrontal cortex of both cerebral hemispheres by electric stimulation of the homologous contralateral cortical region. Fatigability of interhemispheric responses recorded in the right hemisphere exhibited greater amplitude increments to double‐shock stimulation. No significant differences in rheobase, chronaxie, and threshold current were observed when comparing responsiveness of both prefrontal cortices to single stimuli. Results suggest that brain lateralization in the rat could depend, in part, on synaptic functional asymmetries of the prefrontal cortex.
Characterization of the distinctive neurofilament subunits of the soma and axon initial segments in the squid stellate ganglion
Authors: Tytell M. , Pant H. C. , Gainer H. , Hill W. D.
Published: January 2005

The stellate ganglion, which gives rise to the giant axons of the squid, was dissected into two parts, one containing primarily cell bodies and the other axon initial segments. A neurofilament protein‐enriched extract of each was prepared and compared biochemically and immunochemically with an axoplasmic neurofilament preparation and with the glial sheath that surrounds the axons. Both parts of the ganglion lacked the 220 kDa subunit of axoplasmic neurofilaments (NFs). However, they did contain a protein of about 190 kDa that reacted with the Pruss anti‐inter‐mediate filament antibody (aIFA; Pruss et al.: Cell 27:419–428, 1981), but not with a phosphorylationdependent NF antibody (Cohen et al.: I Neurosci 7: 2056–2074, 1987). Dephosphorylation of the axoplasmic NF 220 Vielded a product that comigrated on twodimensional (2D) gel electrophoresis with the 190 kDa ganglion protein, suggesting that the latter represented the incompletely phosphorylated precursor of NF220. The major low molecular weight aIFA‐reactive species in the ganglion preparation was a polypeptide of about 65kDa. A relatively small quantity of that polypeptide was also found in axoplasm and it comigrated in 2D gels with an aIFA‐reactive polypeptide from the glial sheath. These results indicate that the site of modification of the 190 kDa NF precursor to the 220 kDa axonal form is probably at the point where the axon initial segments leave the ganglion, which is several mm distal to its site of synthesis in the cell body. Furthermore, the filament network of the axoplasm and possibly the cell bodies includes a gliallike intermediate filament protein in addition to the NF protein subunits.
Stimulation of adrenergic development in neural crest cultures by a reconstituted basement membrane‐like matrix is inhibited by agents that elevate cAMP
Authors: Maxwell G. D. , Forbes M. E.
Published: January 2005

Previous work (Maxwell and Forbes: Development 101:767–776, 1987) has shown that an overlay of reconstituted basement membrane‐like (RBM) gel dramatically increased the number of catecholaminepositive (CA+) cells which differentiated in neural crest cultures. We report here that this increase was inhibited when cultures were grown for 7 days in the presence of agents that elevate cAMP, such as 8‐bromo‐cAMP and 3‐isobutyl‐1‐methylxanthine. The action of 8‐bromo‐cAMP was dose dependent with a half‐maximal effect at about 50 μM. The development of CA+ cells was dramatically reduced when 8‐bromo‐cAMP was present from days 0–4 in vitro, but was relatively unaffected if 8‐bromo‐cAMP was present from days 4–7 in vitro. The development of tyrosine hydroxylase immunoreactive cells was also inhibited by 8‐bromo‐cAMP. The addition of 8‐bromo‐cAMP increased the number of melanocytes and resulted in either no change or only modest reductions in the number of E/C8 and neurofilament immunoreactive cells, indiciting that the effect on CA+ cell ontogeny was selective. In contrast to the effectof 8‐bromo‐cAMP, addition of 8‐bromo‐cGMP did not inhibit CA+ cell development in the presence of the RBM gel nor did it stimulate CA+ cell development in the absence of the RBM gel overlay. Our results suggest that cAMP may be an important regulator of phenotypic expression in at least some neural crest cell lineages. In particular, these observations provide evidence that agents that elevate cAMP levels can act to prevent an essential part of the normal cellular response leading to the normal cellular response leading to the appearance of CA+ phenotype in neural crest cultures.
Neurite outgrowth promoting activity of G4 and its inhibition by monoclonal antibodies
Authors: Chang S. , Rathjen F. G. , Raper J. A.
Published: January 2005

The chick G4 molecule is a cell surface glycoprotein which is a member of the L1/NILE/NgCAM/8D9 group of neural cell adhesion molecules. Polyclonal antisera against G4 have been shown to decrease sympathetic neurite outgrowth on sympathetic axons and to de‐bundle retinal axons growing on a tectal membrane substrate. We have extended the specificity of these results by showing that a panel of monoclonal antibodies against G4 is also effective in reducing sympathetic neurite outgrowth on sympathetic axons. Furthermore, purified G4 adsorbed onto an inert substrate promotes extensive neurite outgrowth. Monoclonal antibodies to G4 completely inhibit the activity of purified G4. These data show that G4 is a cell surface neurite outgrowth promoting molecule, a function which was first suggested by antibody perturbation experiments and now is confirmed directly.