bluuebunny/crossref_metadata_2025 huggingface dataset:
Modulation of the neurofibromatosis type 1 gene product, neurofibromin, during Schwann cell differentiation
Authors: Gutmann D. H. , Tennekoon G. I. , Cole J. L. , Collins F. S. , Rutkowski J. L.
Published: January 2005

Neurofibromin, the product of the neurofibromatosis type 1 (NF1) gene, is a ∼250 kDa protein expressed predominantly in cortical neurons and oligodendrocytes in the central nervous system (CNS) and sensory neurons and Schwann cells in the peripheral nervous system (PNS). To gain insight into the biological role of neurofibromin in Schwann cells, the modulation of NF1 gene expression in a Schwann cell line (MT4H1) stimulated to either proliferate or differentiate in response to agents that elevate intracellular cAMP was examined. Untreated cells and cells exposed to mitogenic doses of forskolin (1–10 μM) or 8‐bromo‐cAMP (0.1 mM) expressed low levels of NF1 MRNA and the protein was barely detectable. High doses of forskolin (100 μM) or 8‐bromo‐cAMP (1 mM) induced the expression of both myelin P0 protein and neurofibromin with an identical time course. Although NF1 mRNA levels peaked within 1–6 hr, the rise in neurofibromin was not apparent until 24–48 hr and peaked 72 hr after treatment. P0 and neurofibromin were also coinduced by cell‐cell contact in high density, untreated cultures. Moreover, differentiation initiated by either cAMP stimulation or high density culture conditions was associated with predominant expression of the type 2 NF1 mRNA isoform. In contrast, type 1 NF1 mRNA isoform expression was observed in untreated Schwann cells or those stimulated with mitogenic doses of forskolin or 8‐bromo‐cAMP. A switch from the type 1 neurofibromin that can efficiently down‐regulate p21‐ras to the type 2 isoform with reduced activity may facilitate a p21‐ras signaling pathway associated with Schwann cell difrerentiation. © 1993 Wiley‐Liss, Inc.
Culture model for the study of cerebral malaria: Antibodies from Plasmodium falciparum‐infected comatose patients inhibit the dendritic development of Purkinje cells
Authors: Calvet M.‐C. , Druilhe P. , Camacho‐Garcia R. , Calvet J.
Published: January 2005

The in vitro development of kitten Purkinje cells was inhibited by sera or IgG from cerebral malaria (CM) patients but not by sera from acute non‐complicated malaria (NCM) cases. This inhibitory effect, quantified by computer‐assisted methods, concerned the dendritic (though not the axonal) development and was found to be related to the presence in CM sera of self‐reactive antibodies of the IgG class. These results suggest that protection acquired against the major complication of Plasmodium falciparum malaria may correspond to the repression of an abnormal stimulation of autoreactive B cells. © 1993 Wiley‐Liss, Inc.
Gelatinase B is present in the cerebrospinal fluid during experimental autoimmune encephalomyelitis and cleaves myelin basic protein
Authors: Gijbels K. , Proost P. , Masure S. , Carton H. , Billiau A. , Opdenakker G.
Published: January 2005

Gelatinases in inflammatory demyelinating diseases of the central nervous system (CNS) were studied using actively induced experimental autoimmune encephalomyelitis (EAE) in mice as a model system. Clinical disease scores correlated in time and in intensity with pathology parameters such as cytosis in the cerebrospinal fluid (CSF), inflammatory infiltrates, and demyelination in the CNS. Zymographic analysis was employed to measure gelatinases A and B in the CSF from individual animals. According to their apparent molecular weight (MW), gelatinases A and B appeared with a MW of 65 and 95 kDa, respectively. The 65 kDa form was present in all samples, even in those derived from non‐induced animals, whereas the 95 kDa form was present only in samples from animals developing EAE. The levels of 95 and 65 kDa gelatinase correlated with the CSF cytosis. In vitro digestion of myelin basic protein (MBP) with gelatinase B and analysis of the cleavage products by protein sequence analysis pinpointed two cleavage sites in conserved regions of MBP. Gelatinase production within the CNS may constitute an important pathogenic mechanism for both the disruption of the blood‐brain barrier and the destruction of myelin, as observed in several neuroinflammatory disorders. © 1993 Wiley‐Liss, Inc.
Glycerophosphorylcholine phosphocholine phosphodiesterase activity during the differentiation of glial progenitor cells
Authors: Monge M. , Yuan J. , Cabon F. , Zalc B. , Kanfer J. N.
Published: January 2005

O‐2A progenitor cells were grown in medium containing either 1% or 10% fetal calf serum (FCS) for 4 weeks. The cells in 1% FCS were 75% oligodendrocytes by 3 weeks in culture. The cell population was so overgrown with astrocytes in the 10% medium that an accurate estimate of cell number could not be made. The activities of glycerophosphorylcholine phosphocholine phosphodiesterase (GPC‐PC‐PdE), p‐nitrophenylphosphorylcholine phosphodiesterase (pNPPC‐PC‐PdE), and ceramide UDP galactose galactosyl transferase (CGalT) were barely detectable in the cells grown in 10% FCS. The activities of these 3 enzymes were low in the cells grown in 1% FCS for the first 2 weeks and then all 3 increased manyfold. These observations reinforce the evidence previously accrued showing that these two phosphodiesterase activities (GPC‐PC‐PdE and pNPPC‐PC‐PdE) are markers of oligodendroglial cells as well as myelin. In contrast, glycerophosphorylcholine choline phosphodiesterase (GPC‐C‐PdE) activities were present in cells grown in both 1% and 10% FCS. © 1993 Wiley‐Liss, Inc.
Lipocortin 1 immunoreactivity identifies microglia in adult rat brain
Authors: McKanna J. A.
Published: January 2005

Immunohistochemical localization of two Ca++‐binding proteins, Lipocortin 1 (LC1) and S100‐β, demonstrates two distinct classes of primitive glia in the floor plate of rat embryos. With proper fixation (formalin‐lysine‐periodate‐acetic acid), dendritic glia in the CNS of adult rats also apparently stain for either LC1 or S100‐β in the ratio of 1:3. In order to further distinguish and identify these two glial classes, we have examined their population density, topography, and responses to localized neuron death. Neurons of the ipsilateral thalamus undergo apoptosis following cortical ablation; the contralateral thalamus serves as control. By eight days post‐lesion, the number of LC1 cells in the ipsilateral thalamus has increased >4‐fold, the increase comprising primarily activated phagocytes adjacent to degenerating neurons. The S100‐β glia in the same region are virtual‐ ly indistinguishable from control; but background staining (apparently representing extracellular S100‐β) is increased. Thus, the responses of dendritic LC1 glia resemble those previously described for microglia and are quite different from the astrocytes identified by S100‐β immunoreactivity. Both dendritic and activated forms of LC1 glia stain with the microglial marker, Griffonia simplicifolia iso‐lectin B4. However, before the correspondence of LC1 glia and microglia can be confirmed, two anomalies require resolution: (1) the LC1 glia are greater in number and more evenly distributed than microglia marked with other methods; (2) the dendritic LC1 glia apparently are progeny of primitive glia that form the midline raphe of the embryonic floor plate. The participation of LC1 glia in the removal of CNS debris supports the hypothesis that LC1 plays anti‐inflammatory and/or immunosuppressive roles in phagocytes. © 1993 Wiley‐Liss, Inc.
Orthograde transport and release of insulin‐like growth factor I from the inferior olive to the cerebellum
Authors: Nieto‐Bona M. P. , Garcia‐Segura L. M. , Torres‐Aleman I.
Published: January 2005

Insulin‐like growth factor I (IGF‐I) and its receptor are expressed in functionally related areas of the rat brain such as the inferior olive and the cerebellar cortex. A marked decrease of IGF‐I levels in cerebellum is found when inferior olive neurons are lesioned. In addition, Purkinje cells in the cerebellar cortex depend on this growth factor to survive and differentiate in vitro. Thus, we consider it possible that IGF‐I forms part of a putative trophic circuitry encompassing the inferior olive and the cerebellar cortex and possibly other functionally connected areas. To test this hypothesis we have studied whether IGF‐I may be taken up, transported, and released from the inferior olive to the cerebellum. We have found that 125I‐IGF‐I is taken up by inferior olive neurons in a receptor‐mediated process and orthogradely transported to the cerebellum. Thus, radioactivity found in the cerebellar lobe contralateral to the injection site in the inferior olive was immunoprecipitated by an anti‐IGF‐I antibody, co‐eluted with 125I‐IGF‐I in an HPLC column, and co‐migrated with 125I‐IGF‐I in an SDS‐urea polyacrylamide gel electrophoresis. Time‐course studies indicated that orthograde axonal transport is relatively rapid since 30 min after the injection, radiolabeled IGF‐I was already detected in the contralateral cerebellum. Furthermore, transport of IGF‐i from the inferior olive is specific since when 125I‐neurotensin was injected in the inferior olive or when 125I‐IGF‐I was injected in the pontine nucleus, no radiactivity was found in the contralateral cerebellum. In addition, no specific transport of 125I‐IGF‐I was found in climbing fiber‐deafferented rats or when excess unlabeled IGF‐I was co‐injected with 125I‐IGF‐I. We next studied whether IGF‐I is released by inferior olive neurons. We found that the release of IGF‐I from cerebellar slices of normal rats was significantly greater in response to depolarizing stimuli than that from slices obtained of climbing fiber‐deafferented animals. Indeed, in vitro release of IGF‐I in response to KCI or veratridine was almost completely abolished in the latter. These data suggest that IGF‐I is taken up by inferior olive neurons through IGF‐I receptors and transported to the cerebellum through their axons without any major modification. Moreover, the release of IGF‐I from the cerebellum after depolarization depends on the presence of climbing fiber afferents. Altogether these results indicate that the olivo‐cerebellar pathway is able to take up, orthogradely transport, and release IGF‐I. Since a similar process has been described in the visual system for basic fibroblast growth factor (bFGF), we propose that IGF‐I, bFGF, and possibly other growth factors may constitute afferent trophic signals involved in plastic mechanisms within specific neural circuitries. © 1993 Wiley‐Liss, Inc.
Immediate early gene activation during the initial phases of the excitotoxic cascade
Authors: Walker P. D. , Carlock L. R.
Published: January 2005

Direct brain injections of the N‐methyl‐D‐aspartate receptor agonist quinolinic acid (QA) trigger an excitotoxic cascade characterized by rapid neuronal death and glial/immune cell activation. The present study compared the timing of immediate early gene (IEG; c‐fos, c‐jun, jun‐B, and zif/268) induction with the response of neuronal ranscripts during the first 24 hr of a QA lesion within the rodent striatum. Following QA exposure, IEG mRNA induction periods extended from 30 min to 24 hr. Several characteristics of this prolonged transcriptional response suggest that separate cell populations (neuronal vs. glial) originate individual IEG phases during the first day of the lesion. The first IEG phase was rapid and peaked at 60 min. This initial IEG phase, likely neuronal in origin, was dominated by robust increases in the expression of c‐fos, jun‐B, and zif/268 mRNAs in contrast to small increases in c‐jun expression. A second, delayed IEG phase was initiated after the first hour and extended to 24 hr. This IEG phase was more intense and continued beyond the period of neuronal survival as detected by the loss of neurotransmitter‐specific mRNAs (preprotachykinin, preproenkephalin, and glutamic acid decarboxylase). During this phase, c‐jun mRNA levels coordinately increased with c‐fos. Interestingly, the transcriptional peak of the delayed IEG phase occurred between 4 and 12 hr, the time which corresponded to the rapid decline of neuronal transcripts. The temporal association of a delayed phase of IEG induction, possibly originating in nonneuronal cells, with neuronal death during the acute phases of the excitotoxic cascade suggest a link between glial IEG activation and the spreading cascade of neuronal damage characteristic of excitatory amino acid receptor overstimulation. © 1993 Wiley‐Liss, Inc.
Movement of embryonic chick sympathetic neurons on laminin in vitro is preceded by neurite extension
Authors: Liang S. , Crutcher K. A.
Published: January 2005

Chick sympathetic neurons (E‐9) are capable of moving on a laminin substrate but not on more adhesive substrates in vitro. The effect of laminin is dose‐dependent and reduced by the addition of anti‐laminin antibodies, whereas soluble laminin does not stimulate movement. The onset of neuronal movement is preceded by, and highly correlated with, the onset of neurite formation. The addition of 1,2 dioctanoyl‐snglycerol (DAG), a stimulator or protein kinase C that has been shown to inhibit neurite outgrowth, was found to delay both process formation and neuronal movement but did not affect the correlation between these two measures. These results support the conclusion that laminin stimulates primary neuronal movement in vitro and suggest that the mechanism underlying movement involves process formation followed by “towing” of the cell body by the advancing process. The similarities of this in vitro behavior to that observed in vivo suggest that similar mechanisms may underlie neuronal movement in the developing nervous system as suggested by Morest (Z Anat Entwicklungsgesch 130:265–305, 1970) and Liesi (EMBO J 4:1163–1170, 1985; Exp Neurol 117:103–113, 1992). © 1993 Wiley‐Liss, Inc.
High resolution labeling of cholinergic nerve terminals using a specific fully active biotinylated botulinum neurotoxin type A
Authors: Arribas M. , Blasi J. , Egea G. , Fariñas I. , Solsona C. , Marsal J.
Published: January 2005

We report here on the synthesis and characterization of a fully active biotinylated derivative of the botulinum neurotoxin type A. Different ratios of biotin:botulinum toxin were tested to optimize derivatizing conditions and a ratio of 35:1 was selected for further experiments. The average number of biotin groups per toxin molecule was estimated to be 7.8, occurring at both heavy and light chains, and almost all externally located and easily accessible to recognition by streptavidin. The modified toxin retained its toxicity and its ability to interact with biological membranes. Apart from its suitability for detection in Western blots and in microtiter well plates, biotinylated botulinum toxin proved to be adequate for morphological labeling studies at both light and electron microscopy. Peroxidase histochemistry in cryostat sections of intoxicated rat hemidiaphragm muscles showed a distinct labeling of end‐plates. Electron microscopy studies were performed on the electric organ of Torpedo marmorata using colloidal gold‐conjugated streptavidin for detection. After intoxication of electric organ fragments with the modified toxin, gold labels were found associated with the presynaptic plasma membrane of nerve terminals and with the membrane of synaptic vesicles. Moreover, the distribution of biotinylated botulinum toxin binding sites over the membrane of synaptosomes isolated from the electric organ of Torpedo and their relationship with intramembrane particles were analyzed using the replica‐staining label‐fracture technique. It was found that the toxin is never associated with intramembrane particles. © 1993 Wiley‐Liss, Inc.
Reactive mononuclear phagocytes release neurotoxins after ischemic and traumatic injury to the central nervous system
Authors: Giulian D. , Corpuz M. , Chapman S. , Mansouri M. , Robertson C.
Published: January 2005

Reactive microglia and invading macrophages, Which appear in brain damaged by stroke or trauma, secrete neuron‐killing factors. This release of cytotoxic substances is a delayed process and is not detected until inflammatory cells reach a peak of reactivity by the second day after injury. Proximity to the site of injury and density of mononuclear phagocytes determine in part the amount of neurotoxic activity released by injured tissues. Moreover, drugs that suppress the accumulation of reactive microglia and macrophages also reduce tissue production of neuron poisons. Neurotoxins released by brain inflammatory cells or extracted directly from inflamed tissues are heat‐stable, protease‐resistant molecules <500 daltons with actions blocked by N‐methyl‐D‐aspartate (NMDA) receptor antagonists. These molecules are distinguished from free radical intermediates, bind to cation exchange resins, lack carboxyl moieties, and are separated from excitatory amino acids including glutamate or aspartate and from the NMDA receptor‐mediated toxin quinolinc acid by ion exchange and reverse phase chromatography. Our data suggest that an unrecognized class of neuron‐killing molecules produced by inflammatory cells mediate the delayed neuronal loss associated with stroke and trauma. © 1993 Wiley‐Liss, Inc.
CNS white matter can be altered to support neuronal outgrowth
Authors: Carpenter M. K. , Hassinge T. D. , Whalen L. R. , Kater S. B.
Published: January 2005

Previous work has demonstrated that white matter in the adult mammalian CNS inhibits cell adhesion and neurite outgrowth. This phenomenon has been investigated most recently by culturing neurons on cryostat sections of the adult CNS. Employing this same technique, we have found, in accord with others, that neurons seldom adhere to or grow on central nervous system white matter but will attach and grow on gray matter. In the experiments presented here, embryonic rat hippocampal neurons were grown on cryostat sections from the adult rat CNS, in the presence of brain derived glial cocultures. It was found that the white matter in cryostat sections can be modified by interaction with medium conditioned by brain‐derived glial cells. Neurons plated on sections pretreated by such media show significant increases in both attachment and neurite outgrowth. The activity contained in glial conditioned medium is likely complex in nature. While the majority of the activity can be eliminated by heat treatment and trypsinization, neural adhesion but not neurite initiation is affected by protease treatment. Therefore, cell attachment and neurite outgrowth may be regulated by different factors in the conditioned media. © 1994 Wiley‐Liss, Inc.
Lymphoid tissues induce NGF‐dependent and NGF‐independent neurite outgrowth from rat superior cervical ganglia explants in culture
Authors: Kannan Y. , Stead R. H. , Goldsmith C. H. , Bienenstock J.
Published: January 2005

Induction of neurite outgrowth from superior cervical ganglia (SCG) by rat lymphoid tissues was studied using a tissue culture model. Neonatal rat SCG were cultured with 6–12‐week‐old rat thymus, spleen, or mesenteric lymph node (MLN) explants in a Martrigel layer, in defined culture medium without exogenous nerve growth factor (NGF). SCG were also co‐cultured with neonatal rat heart (as positive control) or spinal cord (SC; as negative control). To determine whether inflammation affects the ability of lymphoid tissues to induce neurite outgrowth, we also examined MLN at various times after infecting rats with Nippostrongylus brasiliensis (Nb‐MLN). In one series of experiments, a single lymphoid tissue explant was surrounded by four SCG at a distance of 1 mm. The extent of neurite outgrowth was determinded by counting the number of neurites 0.5 mm away from each ganglion at several time points. Adult thymus and, to a lesser extent, spleen had strong stimulatory effects on neurite outgrowth from SCG after 12 hr or more in culture. For thymus tissue, this was similar to the positive control heart explants. MLN from normal rats had minimal effect on neurite outgrowth; however, Nb‐MLN showed a time‐dependent enhancement of the neurite outgrowth, maximal at 3 weeks after infection. The relative efficacy of neurite outgrowth induction (heart ≥ thymus ≥ Nb‐MLN ≥ spleen ≥ MLN ≥ SC) was confirmed in a second series of experiments where one SCG was surrounded by three different tissue explants. We then examined the role of 2.5S NGF, a well‐known trophic factor for sympathetic nerves, in the lymphoid tissue‐induced neurite outgrowth. Anti‐NGF treatment of co‐cultures of SCG and heart almost completely blocked the neurite outgrowth. Anti‐NGF also significantly inhibited thymus‐ and spleen‐induced neurite outgrowth, but not as effectively as heart‐induced neuritogenesis (93,80, and 77% inhibition at 24 hr; 86,70, and 68% inhibition at 48 hr for heart, thymus, and spleen, respectively). On the other hand, anti‐NGF inhibited only 8% of neurite outgrowth induced by 3‐week post‐infection Nb‐MLN at 24 hr, and 41% at 48 hr. These data show that several adult rat lymphoid tissues exert neurotrophic/tropic effects. The predominant growth factor in thymus and spleen is NGF, while Nb‐MLN produces factor(s) which is (are) immunologically distinguishable from NGF. These neurotrophic/tropic factors are produced during the reactive lymphoid hyperplasia that forms part of the inflammatory response against the nematode, N. brasiliensis. This suggests the possibility that cytokines produced by lymphocytes or other inflammatory cells may stimulate sympathetic neurite outgrowth in vivo. © 1994 Wiley‐Liss, Inc.
Role for the stem cell factor/KIT complex in schwann cell neoplasia and mast cell proliferation associated with neurofibromatosis
Authors: Ryan J. J. , Klein K. A. , Neuberger T. J. , Leftwich J. A. , Westin E. H. , Kauma S. , Fletcher J. A. , DeVries G. H. , Huff T. F.
Published: January 2005

Schwann cells are the primary cell type in the disfiguring lesions associated with neurofibromatosis type 1 (NF‐1). These lesions also contain abnormally high numbers of mast cells, a cell type which develops in response to stem cell factor. We report here that neonatal and adult rat and human Schwann cells, as well as a transfected rat Schwann cell line and a human Schwannoma line derived from an NF‐1 patient, all produced stem cell factor mRNA and protein. In coculture experiments, surface expression of stem cell factor by neonatal rat Schwann cells was profoundly downregulated by contact with dorsal root ganglion neurites. The receptor for stem cell factor, KIT, was not expressed in normal Schwann cells but was expressed in the human Schwannoma line, suggesting that aberrant KIT expression may form an autocrine loop in certain Schwann cell neoplasias. © 1994 Wiley‐Liss, Inc.
Immunological determinants of nerve growth factor involved in p140trk (Trk) receptor binding
Authors: Nanduri J. , Vroegop S. M. , Buxser S. E. , Neet K. E.
Published: January 2005

Monoclonal anti‐NGF antibodies that specifically inhibit the biological activity of mouse β‐NGF were used to study the structural determinants involved in the interaction of NGF with its receptors gp75LNGFR and Trk. None of the three antibodies–N60, M15, and 27/21–showed any reactivity toward denatured NGF. Three experimental methods–radioim‐munoassay (RIA), enzyme‐linked immunoassay (ELISA), and slot blots–detected no significant cross reactivity between the antibodies and BDNF or NT‐3. RIA showed that M15 and N60 recognize the same or an overlapping antigenic site, but 27/21 recognizes a different epitope. Only 27/21, and not N60 or M15, immunoprecipitated β‐NGF crosslinked to LNGFR receptor. Thus, the epitope recognized by 27/12 does not overlap the LNGFR receptor binding site. N60, M15, and 27/21 all block binding of NGF to Trk in a manner consistent with competitive inhibition. Purified Fab fragments of N60 and M15 gave similar results to the intact antibodies. The other subunits present in the 7S complex of NGF, i.e. the α and γ subunits, competitively inhibited binding of antibodies to β‐NGF. Only the γ subunit inhibited phosphorylation of Trk and biological activity of β‐NGF. These findings suggest that the M15, N60, and 27/21 antibodies bind to a specific site on the surface of NGF where they competitively inhibit binding to the Trk NGF receptor. The region encompassing the N‐terminus, the C‐terminus, and the loop on the surface of β‐NGF containing residues 60–80 is proposed as important for binding to the Trk receptoe. © 1994 Wiley‐Liss, Inc.
Differential expression of Lewisx and sialyl‐Lewisx antigens in fetal human neural cells in culture
Authors: Satoh J. , Kim S. U.
Published: January 2005

Lewisx is a cell‐surface carbohydrate antigen defined by the trisaccharide structure, Galβ 1→4 (Fucα 1→3) GlcNAc. Expression of Lewisx and sialyl‐Lewisx antigens in primary cell cultures isolated from fetal human brains of 12‐15 weeks gestation was investigated by double immunolabelling with antibodies against monomeric Lewisx (4C9), oligomeric Lewisx (FH4), and sialylated oligomeric Lewisx (FH6) antigens and cell type‐specific markers. The monomeric Lewisx antigen was expressed in more than 15% of astrocytes and 100% of oligodendrocytes, whereas it was not identified in neurons or in microglia. The oligomeric Lewisx antigen was undetectable in any cell types, while the sialylated oligomeric Lewisx antigen was expressed in more than 95% of microglia but not in any other cell types. The cell type‐specific expression of Lewisx and sialyl‐Lewisx antigens in fetal human glial cells suggests that these fucose‐containing carbohydrate molecules play roles in intercellular recognition between distinct cell types during the development of the human central nervous system. © 1994 Wiley‐Liss, Inc.
Functional identification of integrin laminin receptors that mediate process outgrowth by human SY5Y neuroblastoma cells
Authors: Choi E. S.‐H. , Rettig W. J. , Wayner E. A. , Srour M. L. , Clegg D. O.
Published: January 2005

Treatment of the human neuroblastoma cell line SY5Y with nerve growth factor (NGF) induces terminal neuronal differentiation of a subpopulation of cells which can be selected by treatment with a DNA synthesis inhibitor. We have examined the interactions of navie (untreated) and NGF‐differentiated SY5Y cells with laminin, and identifid integrin receptors that mediate laminin‐induced process outgrowth. Differentiated cells displayed a greater capacity for process extension, which correlated with increased expression of integrin laminin receptors. Both naive and differentiated cells expressed integrins α1/β1, α2/β1, and α3/β1 but the differentiated population expressed about 5‐fold higher levels of α1/β1 and about 2‐fold nore α2/β1 and α3/β1 on their surface. Function blocking monoclonal antibodies were used to identify integrin receptors mediating process outgrowth. The anti‐α1 monoclonal antibodies were used to identify intergrin receptors mediating process outgrowth. The anti‐α1 moniclonal antibody SR84 was shown to block α1 function and inhibit process outgrowth on laminin. Despite the presence of multiple integrins which have been shown to bind laminin in other cell types, α1/β1 mediated the majority of process outgrowth in both naive and differentiated cells, with a minor role played by α3/β1. These data indicate that α1/β1 function is requried for process outgrowth on laminin by SY5Y cells and suggest that increased expression may be a crucial aspect of neuronal differentiation. © 1994 Wiley‐Liss, Inc.
Proteolipid protein interactions in transfectants: Implications for myelin assembly
Authors: Sinoway M. P. , Kitagawa K. , Timsit S. , Hashim G. A. , Hashim G. A. , Colman D. R.
Published: January 2005

The proteolipid proteins (PLP and DM20) are major constituents of CNS myelin, but how they are delivered to and organized within the oligodendrocyte plasma membrane is incompletely understood. We have expressed both PLP and DM20 singly or together in a host cell line, HeLa. In either DM20 or PLP transfectants, at early time points (24 hours), the expressed proteins are found within intracellular compartments. In DM20 transfectants, the protein is delivered to the plasma membrane by 48 hours. In HeLa cells, PLP remains intracellular when expressed in the absence of DM20; only when it is coexpressed with DM20 is it transported to the plasma membrane. In cotransfectants, PLP can also be localized to organelles involved in both the protein biosynthetic and the endocytic pathways. Since, in HeLa cells at least, the delivery of PLP to the plasma membrane is facilitated by the coexpression of DM20, we suggest that the two proteins interact intracellularly to form a complex. In some PLP/DM20 cotransfectants, the proteolipids are concentrated in regions of cell‐cell contact. The regional accumulation of these proteins at cell‐cell interfaces is highly reminiscent of the behavior in transfected cells of another myelin protein, Po, and certain cadherin polypeptides, both of which have readily demonstrable membrane adhesive properties. Our data suggests that at certain stoichiometric ratios, proteolipids can become stablized at cell surfaces to form adhesive bonds. © 1994 Wiley‐Liss, Inc.
Intracellular transport and sorting of the oligodendrocyte transmembrane proteolipid protein
Authors: Gow A. , Friedrich V. L. , Lazzarini R. A.
Published: January 2005

Delineating the properties and functions of the major central nervous system myelin proteins has been the focus of intensive research for decades. For PLP, this task has been confounded by its unusual properties, the complexity of the cellular membrane in which it resides, and the absence of a functional assay for the protein. The development of new experimental paradigms in which to study PLP may shed fresh light on the properties and functions of this intrinsic membrane protein. In the present communication we have used indirect, double label, immunofluorescence, and confocal microscopy to examine the distribution of PLP in Cos‐7 cells transfected with an expression vector bearing the human PLP cDNA. Our results show that PLP is synthesized in the rough endoplasmic reticulum of transfected cells and passes through the Golgi apparatus to the cell surface. These results are consistent with previous studies showing PLP reaches the cell surface by transport through the secretory pathway. Levels of PLP at the cell surface are modest, most likely because protein deposited in this compartment can be endocytosed and subsequently transported to perinuclear lysosomes. Similar results are reported in the companion communication by Sinoway et al. (J Neurosci Res, 37:551–562, 1994). Using transfected HeLa cells they show that DM20 alone and PLP coexpressed with DM20 assume appropriate conformations for transport to the cell surface.

The presence of PLP in subcellular compartments beyond the endoplasmic reticulum in Cos‐7 cells indicates that the protein achieves a conformation appropriate for transport in the absence of other oligodendrocyte‐specific factors; however, accumulation of large amounts of PLP in the cytoplasmic membrane compartment may require interactions with such glial‐specific factors. Thus, the transfection paradigm described herein should prove a useful tool for investigating the folding and sorting of wild type and mutant forms of PLP as well as its membrane topology and posttranslational processing.
Retinal ganglion cell survival in vitro maintained by a chondroitin sulfate proteoglycan from the superior colliculus carrying the HNK‐1 epitope
Authors: Nichol K. A. , Everett A. W. , Schulz M. , Bennett M. R.
Published: January 2005

We recently reported evidence implicating a superior colliculus‐derived chondroitin sulfate proteoglycan (SCCP) in the trophic support of cultured retinal ganglion cells (Schulz et al., 1990). In the present work we show preparations of the SCCP to be reactive with an antibody (CS‐56) to chondroitin sulfate types A and C and with the HNK‐1 antibody. Reaction with the HNK‐1 antibody allowed us partially to purify the native proteoglycan by immunoaffinity chromatography. HNK‐1 reactive material was further processed by a combination of molecular sieve chromatography in the presence of 4M guanidine HCL followed by anion exchange chromatography to yield a product that migrated electrophoretically as a single band in polyacrylamide gel with an apparent molecular weight of not less than 400k. The SCCP, when added to a fully defined culture medium, maintained the survival of the vast majority (80%) of the ganglion cells over a 16 hr culture period with 86% of these cells showing a profusion of processes; few ganglion cells (10%) survived in the absence of the proteoglycan. Electrophoretic analysis of nonreduced preparations of the molecule did not reveal any low molecular weight silver stained components that may have remained associated with the molecule after guanidine HCL treatment. However, two bands corresponding to molecular weights of around 60 and 80 k were reproducibly obseved on polyacrylamide gels following electrophoresis of the molecule in the presence of β‐mercaptoethanol. Our findings provide further evidence suggesting a role for a chondroitin sulfate proteoglycan carrying the HNK‐1 epitope in the trophic support of central neurones. © 1994 Wiley‐Liss, Inc.
Peripheral myelin protein‐22 expression in charcot‐marie‐tooth disease type 1a sural nerve biopsies
Authors: Hanemann C. O. , Stoll G. , D'Urso D. , Fricke W. , Martin J. J. , Van Broeckhoven C. , Mancardi G. L. , Bartke I. , Müller H. W.
Published: January 2005

Peripheral myelin protein‐22 (PMP22) is expressed in myelinating Schwann cells and shows significant homology to murine growth arest‐specific gene gas3. Charcot‐Marie‐Tooth disease type 1a (CMT1a) is a common hereditary demyelinating neuropathy. Recently it was demonstrated that the gene for PMP22 is duplicated in CMT1a patients. A gene dosage mechanism has been postulated to cause CMT1a. According to this hypothesis, the increase in copy number of PMP22 gene would lead to an elevated expression of PMP22 and thereby cause the demyelinating phenotype of CMT1a. In the present communication we analyzed PMP22 mRNA and protein expression in sural nerve biopsies from CMT1a patients and normal controls. We show that PMP22 mRNA expression in CMT1a is not uniform. We found both elevated as well as normal PMP22 mRNA levels in patients. Interestingly, the highest PMP22 mRNA level was found in the least affected patient. In contrast to the mRNA levels, PMP22 was clearly reduced in all CMT1a patients as shown by immunohistochemistry. Thus the CMT1a phenotype may not be strictly correlated with increased PMP22 mRNA and protein expression. Possible roles of PMP22 in the pathogenesis of CMT1a are discussed. © 1994 Wiley‐Liss, Inc.
Molecular cloning and expression of a δ‐opioid recetpor from rat brain
Authors: Abood M. E. , Noel M. A. , Farnsworth J. S. , Tao Q.
Published: January 2005

We have isolated and characterized a rat δ‐opioid receptor. The deduced amino acid sequence (372 aa) closely resembles the murine δ‐opioid receptor, DOR‐1. In fact, 97% of the amino acid residues are conserved between the two species, while 93% of the nucleic acid residues are identical. A 6 kb mRNA was detected in rat cortex using rat DOR‐1 as a probe. When expressed in COS cells, the clone shows high‐affinity opioid binding with selectivity for δ‐opioids. The rat δ‐opioid receptor cDNA clone will be a useful tool for studying the function of δ‐opioid receptor in rats. Wiley‐Liss, Inc.
(–)‐Deprenyl alters the survival of adult murine facial motoneurons after axotomy: Increases in vulnerable C57BL strain but decreases in motor neuron degeneration mutants
Authors: Oh C. , Murray B. , Bhattacharya N. , Holland D. , Tatton W. G.
Published: January 2005

The effect of (–)‐deprenyl on the survival of axotomized adult murine facial motoneurons was investigated. Previously, (—)‐deprenyl was shown to increase the number of rat facial motoneurons (FMns) surviving after axotomy at postnatal day 14, apparently by compensating for the loss of muscle‐derived trophic factor. In the present study, three different strains of adult mice—A/J, C57BL/6J, and a congenic substrain of the C57BL/6J mice, the C57BL/Mnd mutants—underwent unilateral facial nerve transection. FMns were counted from serial sections taken through the entire length of the facial nuclei ipsilateral and contralateral to the facial nerve transections in animals sacrificed 21 days after axotomy. Subgroups of C57BL/6J and Mnd mutants were treated with either saline or 1.0 mg/kg (—)‐deprenyl for 21 days. Another subgroup of Mnd mutants were treated with the metabolites of (—)‐deprenyl, a mixture of (—)‐amphetamine and (—)‐methamphetamine, at a dosage equimolar to 1.0 mg/kg (—)‐deprenyl.

The number of surviving facial motoneurons in the A/J strain was 90% of unlesioned, control values which supports previous findings that adult FMns receive adequate trophic support and thus can survive loss of muscle‐derived trophic support. In the C57BL/6J strain, the facial motoneuron survival was 35% and (—)‐deprenyl increased the survival to 50.5%. Mnd mutants showed 62.4% survival; however, (—)‐deprenyl decreased the number of motoneurons to 54.9% and amphetamine and methamphetamine treatment further decreased the motoneuron survival to 41.1%.

These findings show that FMns in the Mnd mutants and their parental strain, C57BL/6J mice, show greater vulnerability to axotomy as compared to other adult strains of mice. The vulnerability is similar to that found in early postnatal life. (—)‐Deprenyl increases the survival of the axotomized C57BL/6J FMns but its major metabolites, (—)‐methamphetamine and (—)‐amphetamine, further decrease FMn survival in the C57BL/Mnd mutants, possibly due to the induction of neurotoxic proteins causing programmed neuronal death. The efficacy of (—)‐deprenyl in increasing the survival of damaged neurons would be expected to decrease as dosage increased above the dosage sufficient to induce maximum neuronal rescue (approximately 0.01 mg/kg) but would decrease as the dosage exceeded that necessary to produce toxic concentrations of the metabolites of (—)‐deprenyl (1.0 mg/kg in this study). © 1994 Wiley‐Liss, Inc.
Direct immunocytochemical localization of 5‐hydroxytryptamine receptors in the adult rat spinal cord: A light and electron microscopic study using an anti‐idiotypic antiserum
Authors: Ridet J.‐L. , Tamir H. , Privat A.
Published: January 2005

In the present study, we performed the immunodetection of serotonergic (5‐HT) receptor subtypes in the spinal cord by using an anti‐idiotypic antiserum (TH8) at light and electron microscopic levels. This antibody has been shown to recognize 5‐HT1B, 5‐HT1C, and 5‐HT2 receptor subtypes (Tamir et al.: J Neurochem 57:930–942, 1991). The TH8 immunoreactivity was observed in the dorsal and ventral horns of the gray matter. Light microscopy revealed that small cell bodies located in laminae I–III of the dorsal horn were intensely immunolabeled. A more homogenous and discrete staining was also observed throughout the entire dorsal horn. In the ventral horn, motoneurons were also immunoreactive (IR). Peroxidase deposits were observed as numerous patches covering the motoneuronal surface. Numerous interneurons were moderately and homogeneously immunostained.

With the electron microscope, most of the labeled structures were identified as neurons (dendrites and perikarya) in both the dorsal and ventral horns. In the dorsal horn, immunoreactivity was present in dendrites and neuronal perikarya. A large majority of the immunoreactivity found in dendrites was not associated with synaptic differentiations. Indeed, the dendrites, in which peroxidase deposit was seen, were not locally involved in synapses. Very scarce synaptic varicosities were observed in close apposition with IR dendrites. In the ventral horn, TH8 immunoreactivity was present in dendrites, with an accumulation of peroxidase deposit on the active zone of synapses, facing presynaptic membranes. Both the postsynaptic membrane and the submembrane area were IR. In addition, a few astroglial fine processes were immunostained; most of them were observed in the dorsal horn. Scarce IR astroglial profiles were observed in the ventral horn. These observations show that such an antiserum constitutes a useful tool for the ultrastructural analysis of 5‐HT receptor distribution. Finally, correlation between the immunocytochemical localization of 5‐HT receptor subtypes and the modes of 5‐HT transmission in the spinal cord (wiring and volume transmissions) is discussed in the present report. © 1994 Wiley‐Liss, Inc.
Restricted hypotonic swelling of peripheral nerve myelin in streptozocin‐induced diabetic rats
Authors: Kirschner D. A. , Eichberg J.
Published: January 2005

Experimental diabetic neuropathy includes the non‐enzymatic glycosylation (or glycation) of the major proteins of peripheral nerve myelin. We have used X‐ray diffraction to determine whether such glycation affects myelin membrane structure and interactions in peripheral nerves from experimental diabetic rats. Streptozocin at 60 mg/kg was injected intraperitoneally to induce diabetes; controls were pair‐fed and age‐matched. Animals were sacrificed periodically from 2 weeks to after 1 year. The dissected sciatic nerves were tied off and incubated overnight at room temperature in hypotonic saline of defined pH and ionic strength or in distilled water. Such treatments have been shown to result in systematic changes of myelin period, which can be detected using X‐ray diffraction, and which may indicate alterations in inter‐membrane interactions owing to changes in composition. We observed no differences in repeat periods between control and diabetic nerves at pH 4.0 and 7.4, and ionic strength 0.01, 0.02, 0.06, 0.15, and 0.18; however, we did detect a significant difference (P <.02) in their maximum extent of swelling in distilled water: control nerves showed a period of 292 Å (s.d. 23 Å; n = 12) compared to 272 Å (s.d. 19 Å; n = 11) for diabetic nerves. To determine whether this difference in swelling was due to an alteration in the properties of the apposed, extracellular surfaces of the myelin membranes or to the connective tissue in peripheral nerve, we compared the X‐ray patterns from peripheral nerve myelin isolated by sucrose density gradient centrifugation from sciatic nerves of diabetic and control rats. No difference in the patterns was observed. Since the maximum extent of myelin membrane swelling in peripheral nerves is limited by the collagen, our results suggest that the collagen in the nerves of diabetic rats is less elastic or more densely packed than in the controls, most likely due to N‐glycation‐mediated cross‐linking. © 1994 Wiley‐Liss, Inc.
Multiple interactions of aluminum with neurofilament subunits: Regulation by phosphate‐dependent interactions between C‐terminal extensions of the high and middle molecular weight subunits
Authors: Shea T. B. , Beermann M. L.
Published: January 2005

Exposure of individual purified neurofilament (NF) proteins to AlCl3 alters their electrophoretic properties in a time‐ and concentration‐dependent manner, as visualized by their failure to migrate into SDS gels. Co‐incubation of purified high (NF‐H) and middle (NF‐M) but not low (NF‐L) molecular weight NF subunits prevents this AlCl3‐induced alteration in electrophoretic migration. This latter finding suggested that specific interactions between NF‐H and NF‐M other than filament formation influenced their interaction with AlCl3. Co‐incubation of the 160 kDa α‐chymotryptic cleavage product of NF‐H (corresponding to the highly phosphorylated C‐terminal sidearm domain) with native NF‐M prevented alteration in subunit electrophoretic migration by AlCl3. By contrast, intact, dephosphorylated NF‐H subunits were unable to prevent AlCl3‐induced alteration of native NF‐M electrophoretic migration. Taken together, these findings suggest that phosphate‐dependent interactions between the sidearm extensions of NF‐H and NF‐M diminish the ability of AlCl3 to associate with either subunit in a manner that alters their electrophoretic migration. This interaction of NF‐H and NF‐M sidearms is SDS‐sensitive, while AlCl3‐induced alteration in electrophoretic migration of individual subunits is SDS‐resistant. Addition of SDS to mixtures of NF‐H and NF‐M subunits disrupted the protective effect, and promoted AlCl3‐induced alterations in subunit electrophoretic migration. These findings support and extend the current hypothesis that the ability of aluminum to interact with NF subunits is a function of subunit phosphorylation, assembly, and extent of neurofilament‐neurofilament cross‐linking. © 1994 Wiley‐Liss, Inc.
SR13/PMP‐22 expression in rat nervous system, in PC12 cells, and C6 glial cell lines
Authors: De León M. , Nahin R. L. , Mendoza M. E. , Ruda M. A.
Published: January 2005

SR13/PMP‐22 is a protein that was identified after screening a sciatic nerve cDNA library. Our study focused on comparing the level and pattern of expression of SR13/PMP‐22 protein and RNA. Northern blot analysis revealed that although SR13/PMP‐22 mRNA was present in all nervous tissues and cells studied, levels were at least seven fold higher in the sciatic nerve and the spinal cord. During sciatic nerve postnatal development and maturation, the SR13/PMP‐22 mRNA was detected at 2 days after birth, reached a maximal level at day 24, and decreased to 1/3 of the maximum in adult animals. Nerve transection reduced the level of SR13/PMP‐22 mRNA to less than 5% in the segment distal to the nerve injury. Experiments using in situ hybridization localized the SR13/PMP‐22 mRNA in Schwann cells. Schwann cells present in the vicinity or distal to the nerve cut repressed the signal for the message. In situ hybridization experiments also demonstrated that dorsal root ganglia satellite cells contained the message for SR13/PMP‐22. The SR13/PMP‐22 antisera used in our study showed a complex pattern of staining. As expected, the SR13/PMP‐22 antibody peptide 1 immunoreacted with the sciatic nerve sheath. However, immunocytochemistry of the dorsal root ganglia revealed that the staining was contained in the neuron's cell body and processes and also in satellite cells. We also identified immunoreactive cell bodies and fibers in the spinal cord dorsal horn. Tissue culture studies demonstrated that SR13/PMP‐22 mRNA is induced in NGF treated PC12 but not in C6 glioma cell lines grown under experimental conditions that stimulated cell growth arrest. Our experiments suggest that SR13/PMP‐22 may have some other function(s) in addition to its hypothesized role in peripheral myelination. © 1994 Wiley‐Liss, Inc.
Synthesis of 1,25‐dihydroxyvitamin D3 by rat brain macrophages in vitro
Authors: Neveu I. , Naveilhan P. , Menaa C. , Wion D. , Brachet P. , Garabédian M.
Published: January 2005

Cultured microglial cells were examined for their ability to metabolize 25‐hydroxyvitamin D3 (25‐(OH) D3). Upon exposure to lipopolysaccharide, microglial cells produced a vitamin D metabolite which comigrated with synthetic 1,25‐dihydroxyvitamin D3 (1,25‐(OH)2D3) in two different systems of high performance liquid chromatography. This metabolite had the same affinity as synthetic 1,25‐(OH)2D3 for the chick intestinal 1,25‐(OH)2D3 receptor. Lipopolysaccharide‐stimulated microglial cells incubated with 3 nM of 25‐(OH) D3 synthesized up to 5.76 fmol 1,25‐(OH)2D3/8 × 105 cells/2 hr. Microglial cells stimulated for 48 hr with interferon‐γ also produced a significant amount of 1,25‐(OH)2D3 (4.17 fmol/8 × 105 cells/2 hr). In contrast, levels of 1,25‐(OH)2D3 produced by resting microglial cells were barely detectable. It is concluded that activated brain macrophages may be committed to synthesize 1,25‐(OH)2D3 in vitro. This raises the possibility that activation of microglial cells in vivo may be followed by an increase in the level of 1,25‐(OH)2D3 in the central nervous system (CNS). These results support the emerging concept that the brain constitutes a target tissue for vitamin D metabolites. © 1994 Wiley‐Liss, Inc.