bluuebunny/crossref_metadata_2025 huggingface dataset:
MHC class II‐positive microglia in human brain: Association with alzheimer lesions
Authors: Perlmutter L. S. , Scott S. A. , Barrón E. , Chui H. C.
Published: January 2005

Cells of the mononuclear phagocytic system (MPS) present foreign antigen on their cell surfaces bound to major histocompatibility complex (MHC) class II molecules. Previous studies of normal human brain samples reported MHC class II expression primarily by perivascular MPS cells and white matter microglial cells. Marked increases in MHC class II‐expressing microglia have been shown in many neuropathologic disorders, including Alzheimer's disease (AD). A close morphologic association between these cells and Alzheimer senile plaque β‐amyloid has been demonstrated. The present study used a mixed aldehyde fixative to enhance the localization of MHC class II‐expressing MPS cells in non‐AD and AD brain. Two antibodies against MHC class II (HLA‐DR; LN3), as well as the lectin Ricinus communis agglutinin (which recognizes both ramified and activated microglia) were used for light and electron microscopic analyses. We now report that MHC class II‐expressing ramified microglia are distributed in a uniform reticular array throughout the grey, as well as the white matter in non‐AD cases. In AD cases, immunolabelled cells had the morphology of activated microglia, with darkly stained plump somata and short, thick processes. Microglia clustered around senile plaque amyloid and neurofibrillary tangles (NFT), rather than forming the uniform array characteristic of control tissue. Finally, we report that perivascular MPS cells are found in a morphologic relationship with vascular amyloid identical to that seen between microglial cells and senile plaque β‐amyloid. These data suggest that MHC class II‐expressing cells may be involved in the degradation of NFT‐laden neurons and the posttranslational modification of extracellular‐NFT epitopes. In addition, both parenchymal and perivascular MPS cells are ideally situated to uptake and process the β‐amyloid protein precursor and deposit β‐amyloid on senile plaques, NFT, and the cerebrovasculature. © 1992 Wiley‐Liss, Inc.
Acylation of lysophosphatidylcholine by brain membranes
Authors: Premkumar N. , Sun G. Y. , Macquarrie R. A.
Published: January 2005

Brain microsomes catalyze the acylation of lysophosphatidylcholine (lysoPtdCho) in the presence and absence of added CoA derivatives. The catalytic activity is distributed widely in various subcellular fractions from rat or bovine cerebral cortex as measured by the conversion of 1‐[14C] palmitoyl‐sn‐glycero‐3‐phosphocholine to [14C]PtdCho. Analysis of this latter compound revealed that the dipalmitoyl derivative is the predominant molecular species, which is formed in this reaction by transacylation between two [14C]lysoPtdCho molecules. This lysoPtdCho: lysoPtdCho transacylation reaction was enhanced several‐fold by the addition of oleoyl‐CoA, which also is an effective donor of acyl groups in the acyl‐CoA: lysoPtdCho acyltransferase‐catalyzed reaction. Measurements of the initial velocity of the transacylation reaction were used to determine kinetic constants. Apparent Km values for lysoPtdCho in the presence and absence of oleoyl‐CoA were 29 μM and 104 μM, respectively, and the corresponding maximal velocities were 0.11 and 1.06 nmol min−1 mg−1, respectively. Oleoyl‐CoA at 4 μM produced half‐maximal stimulation of the transacylation reaction. CoA also stimulated the rate of conversion of [14C]lysoPtdCho to [14C]PtdCho, either in the presence or absence of oleoyl‐CoA, with a half‐maximal effect of CoA at 80 μM. These results may be important in understanding the regulation of PtdCho synthesis and the mechanism by which acyl group composition of this compound is controlled. © 1993 Wiley‐Liss, Inc.
Influence of janusin and tenascin on growth cone behavior in vitro
Authors: Taylor J. , Pesheva P. , Schachner M.
Published: January 2005

Janusin and tenascin are glia‐derived, structurally related, extracellular matrix glycoproteins of the J1 family that are expressed in vivo at times and in locations where active neurite outgrowth occurs, but also when the formation or stabilization of cytoarchitectonic boundaries appears to be in operation. To resolve this apparent functional dichotomy, we have studied the behavioral response of growth cones, growing in culture on the permissive substrate laminin, to janusin and tenascin, by video time lapse microscopy. When janusin and tenascin were offered as sharp substrate boundaries, dorsal root ganglion (DRG) and retinal ganglion neuron growth cones avoided growing on these molecules, but were not induced to collapse. On the other hand, when janusin and tenascin were offered, in a mixture with laminin, as uniform substrates, DRG growth cones displayed a collapsed morphology and were able to advance at a faster rate than on laminin alone. In contrast, the outgrowth of retinal ganglion neuron growth cones was completely inhibited under these conditions, underscoring a cell type specificity in the response of growth cones to these molecules. Using several monoclonal antibodies binding to distinct epitopes on the tenascin molecule, we have identified two domains responsible for growth cone repulsion, on epidermal growth factor (EGF)‐like repeats 3‐5 and fibronectin type III homologous repeats 4 and 5. These domains are different from the one previously recognized to be involved in neurite outgrowth on a uniform tenascin substrate. We conclude that both molecules may promote or retard growth cone advance, depending on the spatial expression pattern and the neuronal cell type. © 1993 Wiley‐Liss, Inc.
Prednisolone enhances myogenesis and dystrophin‐related protein in skeletal muscle cell cultures from mdx mouse
Authors: Passaquin A. C. , Metzinger L. , Léger J. J. , Warter J.‐M. , Poindron P.
Published: January 2005

The differentiation of skeletal muscle cells from mdx mice which lack dystrophin expression was examined after glucocorticoid treatment, namely α‐methylprednisolone (PDN). Primary skeletal muscle cell cultures were established from newborn mdx, congenic C57BL/10, and allogenic BALB/C mice. We show that PDN promotes the myogenesis of both mdx‐ and control mice‐derived cultures as determined by (1) the number of myotubes, (2) acetylcholine receptors, and (3) dystrophin and dystrophin‐related protein levels. These results support the hypothesis that PDN could enhance the myogenesis of satellite cells and increase dystrophin‐related protein expression in DMD treated patients. © 1993 Wiley‐Liss, Inc.
Evidence for activation of astrocytes via reactive microglial cells following hypoglossal nerve transection
Authors: Svensson M. , Eriksson N. P. , Aldskogius H.
Published: January 2005

Following peripheral nerve injury, resident microglial cells proliferate and astrocytes undergo hypertrophy, as evidenced, e.g., by an increase in the levels of glial fibrillary acidic protein (GFAP). In a previous study we have shown that infusion of cytosine arabinoside (ARA‐C) into the rat brain blocks the axotomy‐induced proliferation of microglial cells. This experimental approach has been used in the present study in order to explore the issue of whether the reactive microglial cells are mediators of the increased GFAP expression in the hypoglossal nucleus of the rat following axotomy. Quantitative analysis of sections processed for immunocytochemistry or in situ hybridization demonstrated a marked increase in GFAP‐like immunoreactivity and GFAP‐mRNA, respectively, in the ipsilateral hypoglossal nucleus 4 and 7 days after axotomy in control experiments. These increases failed to occur in axotomized animals treated with ARA‐C. Therefore, our data are compatible with the hypothesis that activation of astrocytes following axotomy as measured by increased expression of GFAP and its mRNA is induced secondarily to the microglial response. © 1993 Wiley‐Liss, Inc.
Phagocytosis of myelin by microglia in vitro
Authors: Smith M. E.
Published: January 2005

Previous experiments from this laboratory have shown that peritoneal macrophages in culture will phagocytize myelin. Myelin preopsonized with myelin antibodies is phagocytized to a much greater extent than untreated myelin, indicating that macrophages ingest myelin by an Fc receptor. The present work was undertaken to determine the characteristics of myelin phagocytosis by microglia, the resident macrophages of the central nervous system. Microglia isolated from 4–5 day primary cultures of newborn rat brains were shown to bind and phagocytize myelin labeled in the lipids by 14C‐acetate. Both binding and phagocytosis as shown by the appearance of 14C‐cholesterol ester were greatly increased if labeled myelin was preopsonized with antiserum to myelin basic protein or galactocerebroside. Both preopsonized and untreated myelin were phagocytized more actively by microglia than by peritoneal macrophages under the same culture conditions. Microglia cultured in the presence of GM‐CSF showed slightly increased cholesterol ester production from opsonized myelin, but the effect of GM‐CSF was significantly greater than myelin pretreated with control serum (34% increase) or untreated myelin (154% increase). There was no significant effect of GM‐CSF on myelin phagocytosis by peritoneal macrophages. Cerebrospinal fluid containing immunoglobulin drawn from rabbits with acute EAE also opsonized myelin to increase phago cytosis by microglia, as has been previously shown with peritoneal macrophages. These results indicate that microglia may actively participate in myelin destruction in demyelinating diseases where myelin antibodies or a source of GM‐CSF may be present. © 1993 Wiley‐Liss, Inc.

This article is a US Government work and, as such, is in the public domain in the United States of America.
Protein kinase C‐ε is a developmentally regulated, neuronal isoform in the chick embryo central nervous system
Authors: Mangoura D. , Sogos V. , Dawson G.
Published: January 2005

Protein kinase C (PKC) is expressed as many isoforms and in high quantities in the central nervous system (CNS), which suggests an important role for this enzyme in neuronal development and function. We used specific antibodies to investigate the expression of the known PKC isoforms in extracts from chick major CNS areas during embryogenesis, from day 3 (E3) of incubation to day 1 post‐hatching (P1). PKC‐ε was the predominant isoform and was expressed from E6 onward in all brain regions, except retina (E12 and on). PKC‐α/β and ‐ζ isoforms were expressed at lower levels prior to PKC‐ε expression and throughout embryogenesis. No other isoforms were detected in neural tissue preparations. We then used neural culture systems derived from the chick CNS to study the expression of PKC isoforms in neuroblasts, cortical neurons, and cortical glial cells. Western blotting and immunostaining of neuroblastenriched cultures, derived from E3 CNS, showed only the Ca2+‐dependent PKC‐α/β to be present. Studies on neuronal cultures derived from E6 cerebral hemispheres revealed only the Ca2+‐independent PKC‐ε to be expressed in neurons, as predicted by the developmental studies on tissue homogenates. PKC‐ε immunoreactivity was seen intracellularly in differentiating neurons, regardless of their neurotransmitter phenotypes, and it correlated well with the level of neuronal activity. Furthermore, PKC‐α/β immunoreactivity was verified on glia cells, as the glial lineage emerges in E15 cortical cultures. These data suggest that PKC‐ε expression is associated with the final neuroblast division in neurons, and the correlation of PKC isoform expression and neural cell lineage is discussed. © 1993 Wiley‐Liss, Inc.
Retinoic acid responsive gene product, midkine, has neurotrophic functions for mouse spinal cord and dorsal root ganglion neurons in culture
Authors: Michikawa M. , Kikuchi S. , Muramatsu H. , Muramatsu T. , Kim S. U.
Published: January 2005

Midkine (MK) is the product of a retinoic acid responsive gene and is a member of a new family of heparin‐binding growth factors. Neurotrophic effects of MK were examined using cultured spinal cord and dorsal root ganglion (DRG) neurons derived from fetal mouse. MK, which was added to the culture medium at concentrations of 1–100 ng/ml, promoted survival of both types of neurons approximately 5‐fold after 7 days in culture. For spinal cord neurons, the increased survival was reflected in an increase of choline acetyltransferase activity. MK also promoted neurite extension in spinal cord (2‐fold) and DRG (1.7‐fold) neurons. The survival‐promoting activity of MK to these neurons was comparable to that of basic fibroblast growth factor (bFGF) and leukemia inhibitory factor (LIF). In spite of its significant effects on fetal neurons, MK was ineffective in sustaining survival of DRG neurons derived from postnatal mice. From these results, we conclude that MK is a neurotrophic factor to embryonic spinal cord and DRG neurons, and we propose that MK plays a significant role in embryogenesis of the nervous system. © 1993 Wiley‐Liss, Inc.
Effect of vitamin D Deficiency and 1,25‐dihydroxyvitamin D3 on metabolism and D‐glucose transport in rat cerebral cortex
Authors: Stio M. , Lunghi B. , Lantomasi T. , Vincenzini M. T. , Treves C.
Published: January 2005

We previously demonstrated that feeding rats Steenbock and Black's rickets‐inducing diet produces remarkable changes in the metabolic pattern of the in testinal mucosa, kidney, and liver and in some membrane transport Systems of intestinal mucosa and kidney. 1,25‐Dihydroxyvitamin D3 administration to rachitic rats did not always prove to be effective in restoring normal values.

We have now investigated the effect of 1,25‐dihydroxyvitamin D3 on the levels of some metabolites in rat cerebral cortex, on the activity of some enzymes, and on the transport of 2‐deoxy‐D‐glucose and D‐glucose in synaptosomes. Our experiments were carried out on three rat groups: control, rachitic, and rachitic treated with 1,25‐dihydroxyvitamin D3. The decrease in phosphorus content and the increase in citrate concentration observed in rachitic rat cerebral cortex were corrected by 1,25‐dihydroxyvitamin D3 treatment.

The activity of acetylcholinesterase, glucose‐6‐phosphate dehydrogenase, and acyl phosphatase significantly increased in rachitic rat synaptosomes, as well as NAD+‐dependent isocitrate dehydrogenase in cerebral cortex mitochondria; the administration of 1,25‐dihydroxyvitamin D3 to rachitic rats restored enzyme levels to normal. The transport of 2‐deoxy‐D‐glucose and D‐glucose in rachitic rat synaptosomes was lower than in the control group and returned to control values in consequence of 1,25‐dihydroxyvitamin D3 treatment.

The results reported here support the hypothesis of a participation of 1,25‐dihydroxyvitamin D3 in some aspects of cerebral cortex metabolism.
Endogenous peptide Tyr‐Pro‐Trp‐Gly‐NH2 (Tyr‐W‐MIF‐1) is transported from the brain to the blood by peptide transport system‐1
Authors: Banks W. A. , Kastin A. J. , Ehrensing C. A.
Published: January 2005

Tyr‐W‐MIF‐1 (Tyr‐Pro‐Trp‐Gly‐NH2) is a recently isolated peptide that belongs to a larger family that includes Tyr‐MIF‐1 (Tyr‐Pro‐Leu‐Gly‐NH2) and MIF‐1 (Pro‐Leu‐Gly‐NH2). Despite similarities in structure, Tyr‐MIF‐1 and MIF‐1 can act differently in behavioral, blood‐brain barrier (BBB) transport, and receptor binding Systems. Tyr‐W‐MIF‐1, like Tyr‐MIF‐1, has both Opiate and antiopiate activity, but may be more opiate‐like than Tyr‐MIF‐1. TyrMIF‐1, but not MIF‐1, is transported from brain to blood by peptide transport system (PTS)‐1. PTS‐1 transports mainly Tyr‐MIF‐1 and methionine enkephalin, but does not transport amino acids, peptide fragments of Tyr‐MIF‐1, D‐Tyr‐MIF‐1, or unrelated peptides and proteins. We tested whether Tyr‐WMIF‐1 also was transported across the BBB and, if so, whether PTS‐1 was involved. 125I‐Tyr‐W‐MIF‐1 had a half‐time disappearance from the brain of 22.4 min. This is faster than the efflux occurring with nonsaturable reabsorption ofthe cerebrospinal fluid and, therefore, is consistent with saturable transport, but it is slower than the efflux rate of Tyr‐MIF‐1, suggesting a less robust transport than for Tyr‐MIF‐1. Self‐inhibition with excess unlabeled Tyr‐W‐MIF‐1 confirmed a saturable component, with a dose of 4.2 nmol producing 50% inhibition. Competition studies between radioactively labeled Tyr‐W‐MIF‐1 and unlabeled Tyr‐MIF‐1 and between labeled Tyr‐MIF‐1 and unlabeled Tyr‐W‐MIF‐1 showed that Tyr‐WMIF‐1 is transported by PTS‐1 with Tyr‐MIF‐1 being the preferred ligand. Aluminum and serotonergic agents modulate PTS‐1 activity and they affected Tyr‐W‐MIF‐1 transport in a manner consistent with its mediation through PTS‐1. It is concluded that Tyr‐W‐MIF‐1 is transported out of the brain by a saturable system that is probably PTS‐1, although transport is less robust than that of Tyr‐MIF‐1. © 1993 Wiley‐Liss, Inc.

This article is a US Government work and, as such, is in the public domain in the United States of America.
Serotonin‐containing terminals synapse on septohippocampal neurons in the rat
Authors: Milner T. A. , Veznedaroglu E.
Published: January 2005

Serotonin [5‐hydroxytryptamine (5‐HT)] is thought to be involved in mnemonic functions and dysfunctions possibly by directly contacting neurons in the medicl septal and diagonal band nuclei (i.e., the septal complex) that project to the hippocampal formation. However, there is no cellular substrate for this modulation. Thus, we examined the ultrastructure and synaptic association of 5‐HT‐containing terminals in relation to septohippocampal neurons in the septal complex of the rat brain. Projection neurons were identified by retrograde transport of wheat germ agglutinated apo‐horseradish perodidase conjugated to colloidal gold particles (WANG) following an injection into the ventral hippocampal formation of uneashetized adult rats. After a 1 day survival, setions through the septal complex were labeled with antibodies to 5‐HT immunoreactivity (5‐HT‐I) were observed in close proximity to neurons containing retogardely transported WAHG. By electron microscopy, 5‐HT‐I immunoreacitity (5‐HT‐I) were observed in close proximity to neurons containing retrogadely transported WAHG. By electron microscopy, 5‐HT‐I was found exclusively in axons and axon terminals. Axons were primarily unmyelinated. Terminals with 5‐HT‐I were 0.35–1.2 μm in diameter and contained numerous small, clear vesicles and 0–4 large, dense‐core vesicles. The 5‐HT‐labeled terminals: (1) contacted perikarya and dendrites (220 of 349); (2) were closely apposed to other terminals (25 of 349); or (3) had no neuronal contacts in the plane of section analyzed (104 of 349). The 5‐HT‐labeled terminals formed exclusively symmetric synases on perikarya; some of these perikarya as well as some large dendrites similarly contacted by the 5‐HT‐labeled terminals also contained WAHG affiliated with lysosomes and multivesicular and “sequestration” bodies in the cytoplasm. However, the majority of terminals with 5‐HT‐I formed contacts on the shafts of small unlabeled dendrites (69% of 220); most of these were characterized as either asymmetric synapses or appositions not separated by astrocytes in the plane of section analyzed. We conclude that 5‐HT‐containing terminals in the rat septal complex: (1) directly modulate septohippocampal and other neurons through symmetric (potentially inhibitory) synapses on soma and proximal dendrites; and (2) form primarily asymmetric (potentially excitatory) synapses with distal (small) dendrites from neurons of unidentified origin. These findings suggest that serotonin may affect learning and memory through modulation of septal efferents to the hippocampal formation and may have direct relevance to the neuropathological basis for Alzheimer's disease. © 1993 Wiley‐Liss, Inc.
Analysis of the human MBP promoter in primary cultures of oligodendrocytes: Positive and negative cis‐acting elements in the proximal MBP promoter mediate oligodendrocyte‐specific expression of MBP
Authors: Wrabetz L. , Shumas S. , Grinspan J. , Feltri M. L. , Bozyczko D. , McMorris F. A. , Pleasure D. , Kamholz J.
Published: January 2005

Since the regulation of myelin basic protein expression depends primarily on the initiation of transcription, we analyzed the 5′ flanking region of the human myelin basic protein gene in transient transfection studies in primary cultures of developing oligodendrocytes. We demonstrated that 149 base pairs 5′ of the initiation of transcription was sufficient to direct oligodendrocyte‐specific expression of myelin basic protein. The capsite of the fusion transcript was identical with that of the endogenous myelin basic protein transcript, and chloramphenicol acetyl transferase reporter gene expression was restricted to oligodendrocytes in these cultures. Within this 149 base pair region, one distal, negative cis‐acting segment, containing a consensus nuclear factor I site, and one proximal, positive cis‐acting segment were identified. The distal segment behaved more negatively in Cos‐7 cells than in oligodendrocytes, reducing expression to background levels. Furthermore, these functionally important cis‐acting segments bound oligodendrocyte nuclear proteins in a pattern differing from other cells, including Cos‐7 cells. Interestingly, the distal segment increased heterologous SV40 promoter activity in oligodendrocytes but had no effect on the SV40 promoter in Cos‐7 cells. We conclude that the functionally negative distal segment may mediate oligodendrocyte‐specific expression of MBP by restricting its expression in other cells. These experiments strongly support using primary cultures of oligodendrocytes for analyzing the myelin‐specific promoters. © 1993 Wiley‐Liss, Inc.
Platelet‐activating factor is a messenger in the electroconvulsive shock‐induced transcriptional activation of c‐fos and zif‐268 in hippocampus
Authors: Marcheselli V. L. , Bazan N. G.
Published: January 2005

Platelet‐activating factor (PAF, 1‐O‐alkyl‐2‐acetyl‐sn‐glycero‐3‐phosphocholine), undetectable in resting neural tissue, accumulates in brain during seizures. A hetrazepine, BN‐50730, is shown here to displace [3H]PAF‐specific binding from microsomal, but not from synaptosomal membranes, indicating selectivity for a high affinity intracellular binding site. Rats pretreated with BN‐50730 by intraperitoneal or intracerebroventricular injection exhibited an inhibition of the electroconvulsive shock (ECS)‐induced expression of c‐fos and zif‐268 in hippocampus. A much more pronounced, dose‐dependent inhibition of ECS‐induced zif‐268 mRNA in hippocampus by intracerebroventricular injection of of BN‐50730 was observed. It is concluded that, in the hippocampus, PAF IS A MEDIATOR OF THE EXPRESSION OF zif‐268 and, to a lesser extent, c‐fos through an intracellular specific binding site. Thus, PAF may be a messenger in signal regulated zinc‐finger transcription factors, and in other immediate‐early genes involved in long‐term synaptic plasticity changes. © 1994 Wiley‐Liss, Inc.
The long‐term down‐regulation of dihydropyridine receptors by bay K 8644 in PC12 cells
Authors: Nikodijevic B. , Nikodijevic‐Kedeva D. , Oshima M. , Paige B. , Guroff G.
Published: January 2005

Treatment of PC12 cells with Bay K 8644 for 12 hr or more leads to an almost 80% decrease in the subsequent ability of Bay K 8644 to stimulate the uptake of radioactive calcium into the cells. This effect is a property of the s(−)isomer of Bay K 8644; pre‐treatment with the R(+)isomer, now known to be a calcium channel blocker, has the opposite effect. This treatment is specific in that is does not intergfere with the stimulation of calcium uptake by potassium, ATP, or nerve growth factor. Such treatment is accompanied by a 90% decrease in the ability of Bay K 8644 to stimulate the release of norepinephrine. The characteristics of the binding of [3H]isradipine to control and to treated cells indicates that the decrease in the effect of dihydropyridines is accompanied by a marked decrease in the number of dihydropyridine binding sites with no apparent change in the affinity of the remaining sites. The continued ability of depolarizing levels of potassium to stimualte calcium uptake and the induction of the protooncogene c‐fos in Bay K 8644‐treated cells indicates that the L‐type calcium channels are still intact, but are simply unresponsive to dihydropyridine agonists. © 1994 Wiley‐Liss, Inc.

This article is a US Government work and, as such, is in the public domain in the United States of America.
T cell receptor Vβ gene usage in the recognition of myelin basic protein by cerebrospinal fluid‐ and blood‐derived T cells from patients with multiple sclerosis
Authors: Chou Y. K. , Burenafe A. C. , Dedrick R. , Morrison W. J. , Bourdette D. N. , Whitham R. , Atherton J. , Lane J. , Spoor E. , Hashim G. A. , Offner H. , Vandenbark A. A.
Published: January 2005

Because of its proximity to the central nervous system, the cerebrospinal fluid (CSF) represents an important source of T cells that potentially could mediate putative autoimmune diseases such as multiple sclerosis (MS). To overcome the low CSF cellularity, we evaluated culture conditions that could expand CSF T cells, with a focus on the expression of T‐cell receptor Vβ genes utilized by T cells specific for the potentially encephalitogenic autoantigen myelin basic protein (BP). Expansion of “activated” CSF cells with IL‐2/IL‐4 plus accessory cells optimally retained BP‐responsive T cells that over‐expressed Vβ1, Vβ2, Vβ;5, or Vβ;18, compared to expansion using supernatants from PHA‐stimulated blood cells, or anti‐CD3 antibody that led to different V gene bias and rare reactivity to BP. Sequential evaluation of paired CSF and blood samples from a relapsing remitting MS patient indicated that BP‐reactive T cells were present in CSF during the period of clinical activity, and the pattern of BP recognition in CSF was partially reflected in blood, even after CSF reactivity had dissipated during remission. Over‐expressed Vβ genes were not always constant, however, since in three sequential evaluations of a chronic progressive MS patient, Vβ genes over‐expressed in the first BP‐reactive CSF switched to a different Vβ gene bias that was present in the second and third CSF samples. Blood samples reflected each pattern of CSF Vβ gene bias, but retained the initial bias for at least 4 months after its disappearance from CSF. These data indicate that selective expansion of IL‐2/Il‐4‐responsive CSF cells favors growth of the BP‐reactive subpopulation, and, in a limited number of patients studied, reflected clinical disease activity. In comparison, blood T cells provided a partial but longer lasting reflection of the CSF BP reactivity and Vβ gene bias. © 1994 Wiley‐Liss, Inc.
Leukemia inhibitory factor prevents the death of axotomised sensory neurons in the dorsal root ganglia of the neonatal rat
Authors: Cheema S. S. , Richards L. , Murphy M. , Bartlett P. F.
Published: January 2005

Leukemia inhibitory factor (LIF) has several characteristics of a neurotrophic factor for sensory neurons. Here we have investigated whether LIF also supports the survival of axotomised sensory neurons in vivo. Newborn rat pups received a unilateral sciatic nerve transection and the injury site was treated with gelfoam soaked in phosphate buffered saline (PBS), nerve growth factor (NGF), or LIF. Neuronal nucleoli in the L5 dorsal root ganglia were counted, appropriate corrections applied, and the resultant neuronal loss expressed as a percentage of the contralateral intact side. In animals where LIF was administered neuronal loss was significantly reduced: 2 days after LIF treatment neuronal loss was 19.5% compared to 43% in PBS‐treated animals; 3 days after LIF treatment neuronal loss was 20.4% compared to 40.2% in PBS‐treated animals; however, 7 days after LIF treatment there was no significant reduction in the number of neurons lost. The degree of rescue of sensory neurons in vivo by LIF was found to be similar to NGF, which was not surprising as both factors supported the survival of a similar population of sensory neurons in vitro. Rescue was not observed when LIF‐containing gelfoam was placed away from the axotomised nerve, suggesting that LIF's action may be associated with its retrograde transport or direct signalling at the site of nerve injury. © 1994 Wiley‐Liss, Inc.
Expression of the neural cell adhesion molecule and polysialic acid during early mouse embryogenesis
Authors: Probstmeier R. , Bilz A. , Schneider‐Schaulies J.
Published: January 2005

The expression of the neural cell adhesion molecule (N‐CAM) and α 2–8 linked polysialic acid (PSA), which is believed to be predominantly expressed on N‐CAM, was investigated during early embryonic development of the mouse (embryonic days 7.5 to 10.0). By immunocytochemistry, in tissue sections, N‐CAM and PSA were not detectable at embryonic day 7.5 but were expressed in the prominent body regions such as somites, unsegmented mesoderm, developing heart, and neuroectoderm at embryonic day 8.0 N‐CAM and PSA immunoreactivities were always predominantly associated with the plasma membrane. No tissue could be detected which was positive for PSA but negative for N‐CAM. In Western blot analysis of whole embryos, by contrast, only the lightly sialylated and PSA‐negative 180 and 140 kD isoforms of N‐CAM were present at embryonic day 8.0 and strong expression of PSA‐bearing, heavily sialylated N‐CAM was not detectable before embryonic day 10.0. In Western blot analysis of N‐CAM immunoaffinity purified from whole embryos and digested with neuraminidase as well as in Northern blot analysis, the 120 kD isoform of N‐CAM or its corresponding mRNA were not expressed in detectable amounts during the time period investigated. © 1994 Wiley‐Liss, Inc.
Tyrosine phosphorylation of glycoproteins in the adult and developing rat brain
Authors: Soulliere J. , Bissoon N. , Khurgel M. , Gurd J. W.
Published: January 2005

The tyrosine phosphorylation of glycoproteins in the adult and developing rat brain was investigated. Immunoblotting with anti‐tyr(P) antibodies identified a glycoprotein with an apparent Mr of 180,000 (GP180) as the major tyrosine‐phosphorylated protein in the concanavalin A (con A)‐binding fraction prepared from forebrain homogenates. This glycoprotein had the same electrophoretic mobility as the postysynaptic density (PSD)‐associated glycoprotein PSD‐GP180. Tyrosine‐phosphorylated GP180 was enriched 24‐fold in isolated PSDs relative tohomogenates. Digestion with endoglycosidase F/N‐glycosidase F demonstrated that GP180 present in total homogenates and PSD‐GP180 present in total homogenates and PSD‐GP180 present in isolated PSDs contained similar amounts of N‐linked oligosaccharide suggesting that they are the same glycoprotein. The tyrosine phosphorylation of GP180 in homogenates varied between brain regions with the highest levels occurring in cortical areas and the amygdala and low or undetectable amounts being present in hindbrain regions. Incubation of homogenates with adenosine triphosphate (ATP) resulted in the tyrosine phosphorylation of GP180 in all regions examined except the cerebellum and identified a second con A‐binding glycoprotein, GP110, which was phosphorylated on tyrosine. GP180 was not phosphorylated on tyrosine following the incubation of cerebellar homogenate, synaptic membranes, or PSDs with ATP. Tyr(P)‐GP180 was not detected prior to the onset of synaptogenesis, increased in parallel with the formation of synapses during the first 4 weeks of postnatal development of the frontal cortex and hippocampus, and then decreased 50–60% to adult levels. The results suggest that GP180 corresponds to the PSD glycoprotein PSD‐GP180 and are consistent with a role for this glycoprotein in synaptic development and signal transduction at the synapse. © 1994 Wiley‐Liss, Inc.
Amyloid precursor protein is enriched in radial glia: Implications for neuronal development
Authors: Trapp B. D. , Hauer P. E.
Published: January 2005

The amyloid precursor protein (APP) is a transmembrane glycoprotein and the source of the amyloid that accumulates in brains of individuals with Alzheimer's and other neurodegenerative diseases. Although its function is unknown, previous in vitro studies indicate that APP can facilitate neuronal cell adhesion and neuronal survival. If APP has a similar function in vivo, it should be expressed in fetal brain. To investigate this possibility, the present study determined the cellular distribution of APP in fetal and postnatal mouse brain. Immunocytochemistry and confocal microscopy detected abundant APP immunoreactivity in radial glial cells in fetal and early postnatal mouse brain. In mature mouse brain, APP was detected in neuronal perikarya and dendrites but not in astrocytes. These results support a functional role for APP during radial glia‐mediated neuronal histogenesis. © 1994 Wiley‐Liss, Inc.
Isolation and partial characterization of a cell‐surface heparan sulfate proteoglycan from embryonic rat spinal cord
Authors: Guiseppetti J. M. , McCarthy J. B. , Letourneau P. C.
Published: January 2005

Cell‐surface heparan sulfate proteoglycans (HSPGs) are potential mediators of neuronal cell adhesion, spreading, and neurite outgrowth on various extracellular matrix molecules. One possible site of HSPG attachment is a heparin binding domain of fibronectin, which is present in the synthetic peptide FN‐C/H II. In this study, HSPGs extracted from embryonic rat spinal cord by detergent were purified by ionexchange chromatography, gel filtration, and affinity chromatography on an agarose column coupled with FN‐C/H II conjugated to ovalbumin (OA). Heparitinase treatment of the iodinated HSPG fraction led to the appearance of a major protein core with a molecular size of 72 kDa, as determined by reducing SDS‐PAGE. The intact proteoglycan has a molecular size of approximately 150–165 kDa, containing heparan sulfate glycosaminoglycan chains of about 10–15 kDa. Anti‐HSPG antibodies recognized the 72 kDa core protein by immunoblotting, and stained the surface of spinal cord neurons, oligodendrocytes, and a subset of astrocytes. These results identify a cell‐surface HSPG that may mediate neuron‐substratum or neuron‐glia interactions in embryonic central nervous system. © 1994 Wiley‐Liss, Inc.
Dystrophin‐related protein is found in the central nervous system of mice at various developmental stages, especially at the postsynaptic membrane
Authors: Kamakura K. , Tadano Y. , Kawai M. , Ishiura S. , Nakamura R. , Miyamoto K. , Nagata N. , Sugita H.
Published: January 2005

Dystrophin deficiency is known to be the cause of X‐linked Duchenne muscular dystrophy (DMD). A recently cloned B3‐cDNA shares 80% homology with the C‐terminus and actin‐binding portion of dystrophin. This autosome‐derived gene product is called dystrophin‐related protein (DRP). DRP is known to exist in fetal muscles even in mdx mice, an animal model for X‐linked DMD, but not in mature mouse muscles. We raised a polyclonal antibody against a B3‐unique amino acid sequence (Ab‐LDP) and investigated the existence and distribution of DRP in the central nervous system (CNS) tissues of (mdx) and normal control B10 mice at various stages of development using immunoblotting and immunohistochemical methods. The former shows that DRP exists in the CNS of both B10 and mdx mice, regardless of the developmental stage, with the exception that the 420 kDa DRP band of the 15‐day fetus is faint. In immunohistochemical studies, the choroid plexus, some neurons, glial cells, pia mater, and blood vessels were stained with Ab‐LDP. Staining intensity did not differ between B10 and mdx mice of between developmental stages except that the 15‐day fetus stained only faintly. This is in contrast of the results obtained for muscles in which DRP localized to muscle membrane in embryo decreases and is assembled at the neuromuscular junction in adults. In addition, an electron microscopic study on the cerebral cortex from adult B10 mice was also performed and revealed Ab‐LDP staining of the postsynaptic membrane of dendrite and the rough endoplasmic reticulum of neurons. This is a new finding and is of interest because DRP is expressed at the neuromuscular junction and its localization is similar to that of the acetylcholine receptor. Wiley‐Liss, Inc.
Depolarizing agents and tumor necrosis factor‐α modulate protein phosphorylation in oligodendrocytes
Authors: Soliven B. , Takeda M. , Szuchet S.
Published: January 2005

Membrane depolarization and changes in ionic fluxes have been implicated in the signaling mechanisms between neurons and glial cells. We report here that K+‐induced depolarization of cultured ovine oligodendrocytes (OLGs) decreases the phosphorylation of myelin basic protein (MBP) and 2′3′‐cyclic nucleotide phosphohydrolase (CNPase). Membrane depolarization and decrease in phosphorylation of MBP and CNPase can also be elicited by inhibition of the inward rectifier with Ba2+ but not by inhibition of outward K+ channels with 4‐aminopyridine or tetraethylammonium. These findings demonstrate that modulation of K+ currents can influence phosphorylation states of OLG proteins. Tumor necrosis factor‐α (TNF‐α), an immune peptide implicated in autoimmune demyelinating diseases, also inhibits the phosphorylation of these proteins. In contrast to elevated [K+]0, TNF‐α does not decrease the stimulatory effect of protein kinase C activators or phosphatase inhibitors on MBP and CNPase phosphorylation, suggesting that depolarizing agents and TNF‐α act via distinct mechanisms. We postulate that the presence of elevated extracellular K+ and/or cytokines under certain pathological conditions can perturb OLG function by altering the phosphorylation states of their proteins and perhaps affect myelin maintenance, contributing to demyelination. © 1994 Wiley‐Liss, Inc.
Isolation, characterization, and expression of cDNA clones that encode rat UDP‐galactose: Ceramide galactosyltransferase
Authors: Stahl N. , Jurevics H. , Morell P. , Suzuki K. , Popko B.
Published: January 2005

UDP‐galactose:ceramide galactosyltransferase (CGT) (EC. 2.4.1.62) catalyzes the final step in the synthesis of galactocerebroside (GalC), a glycosphingolipid found in high amounts in the myelin sheath. Here, the isolation of rat CGT specific cDNA clones is reported. The CGT sequence contains an open reading frame of 1,623 bp which predicts a protein of Mr 61,126 Da. In transfection experiments the cDNA was found to confer CGT activity of Chinese hamster ovary cells. In rat brain the developmental expression pattern of CGT mRNA was similar to the myelination profile, whereas the sciatic nerve contained high amounts of CGT message over a long developmental period. CGT mRNA expression in the sciatic nerve was found to drop substantially following nerve injury and recover slowly when compared to the expression of mRNAs specific for the predominant myelin‐specific proteins. The absolute amounts of CGT message in sciatic nerve and brain were found to be comparable to those that encode the structural proteins of myelin. Except for low amounts in the kidney, the CGT mRNA was not detected in other tissues examined. Southern blot analysis revealed that the CGT protein is likely encoded by a single, relatively large gene. © 1994 Wiley‐Liss, Inc.
Astrogliosis in culture: III. Effect of recombinant retrovirus expressing antisense glial fibrillary acidic protein RNA
Authors: Ghirnikar R. S. , Yu A. C. H. , Eng L. F.
Published: January 2005

Injury to the central nervous system (CNS) either from trauma or due to demyelinating/degenerating diseases results in a typical response of astrocytes, termed astrogliosis. This reaction is characterized by astrocyte proliferation, extensive hypertrophy of nuclei, cell body, and cytoplasmic processes and an increase in immunodetectable glial fibrillary acidic protein (GFAP). GFAP accumulation may cause a physical barrier preventing the reestablishment of a functional environment. Our studies have aimed at modulating astrogliosis by inhibiting or delaying GFAP synthesis in damaged and reactive astrocytes. The present study investigates the use of a recombinant retrovirus expressing antisense GFAP RNA in controlling the response of mechanically injured astrocytes. A 650 bp fragment from the coding region of mouse GFAP cDNA was cloned in the antisense orientation under the control of long terminal repeat (LTR) promoter of Moloney murine leukemia virus. Increase in GFAP as detected by immunocytochemical staining in injured astrocytes was inhibited by treatment with retrovirus expressing antisense GFAP RNA. Also, astrocytes at the site of injury in these scratched cultures did not show cell body hypertrophy compared to control cultures. These observations demonstrate that the increase in GFAP at the site of injury can be inhibited using retroviral treatment and indicate the potential of retrovirus‐mediated gene transfer in modulating scar formation in the CNS in vivo. These studies also shed light on the role of GFAP in maintaining the morphology of astrocytes. © 1994 Wiley‐Liss, Inc.
Glial cells derived from aged mouse brain in culture display both mature and immature astrocytic phenotypes
Authors: Vernadakis A. , Kentroti S.
Published: January 2005

In earlier studies, we established glial cell cultures derived from aged (18‐month‐old) mouse cerebral hemispheres (MACH) and have maintained them frozen at various passages. These cultures were characterized immunocytochemically and consist of: 5% oligodendrocytes (GalC+), 75% astrocytes‐type 1 (GFAP+ only), 15% astrocytes‐type 2 (GFAP+ + A2B5+), and 5% progenitor glial cells (A2B5+ only). In the present study, we isolated colonies from MACH passage 29 cultures and also colonies from MACH passage 19 transfected with the gene for SV40 large T antigen and further subcultured for 8 passages. Using double‐staining immunocytochemistry, we found in non‐transfected MACH passage 19 colonies consisting primarily of cells exhibiting only vimentin‐positive staining and are considered to be immature glioblasts; colonies consisting primarily of cells exhibiting GFAP+ + vimentin+ which are considered to be astrocytes at an intermediate stage of maturation; and colonies consisting predominantly of cells exhibiting GFAP+ only which are considered to be mature astrocytes. In contrast, colonies isolated from transfected MACH cultures consisted primarily of vimentin+ cells. In conclusion, astrocytes in cultures derived from aged brain continue to be variable as they are during development. However, their response to the microenvironment may differ during development and during aging. Thus, the availability of clones of mature and immature astrocytes offers the opportunity to study neuron‐glia interactions and the role of mature and immature astrocytes in neuronal aging and regeneration. © 1994 Wiley‐Liss, Inc.
Light‐dependent regulation of the transcriptional activity of the mammalian gene for arrestin
Authors: McGinnis J. F. , Austin B. J. , Stepanik P. L. , Lerious V.
Published: January 2005

The purpose of this investigation was to determine if the cyclic variations in the amount of arrestin mRNA that occur in the mouse retina during the light/dark cycle are controlled by light, a circadian clock, or both. In this study, an experimental paradigm was designed and used to distinguish among these molecular mechanisms. The data show that the initiation and the maintenance of an increased rate of syntheses of arrestin mRNA requires light and is not dependent on the time of day. Therefore the changes in transcriptional activity of the mouse arrestin gene are controlled by light, independent of circadian clock. The mechanism for this light‐dependent regulation of gene transcription in the retina is distinct from the light‐dependent increases in mRNA concentrations of early response genes which occur in the suprachiasmatic nucleus and a comparative analysis of such changes in these different regions of the CNS may lead to the identification of the molecular relationship between light‐ and circadian‐dependent gene regulation. © 1994 Wiley‐Liss, Inc.
p75 nerve growth factor receptor modulates p140trkA kinase activity, but not ligand internalization, in PC12 cells
Authors: Kahle P. , Barker P. A. , Shooter E. M. , Hertel C.
Published: January 2005

The biological activity of nerve growth factor (NGF) has been shown to be mediated by the p140trkA receptor tyrosine kinase, while the role of the p75 NGF receptor (p75NGFR) is still unresolved. Here we have investigated the relative contribution of p140trkA and p75NGFR to early consequences of NGF binding: ligand internalization, p140trkA autophosphorylation, and tyrosine phosphorylation of Shc, phospholipase Cγ‐1 (PLCγ‐1), and extracellular signal‐regulated kinases (ERKs). It was found that NGF internalization was neither prevented by blocking p140trkA activity using the protein kinase inhibitors methylthioadenosine, staurosporine, and K‐252a, nor by inhibiting NGF binding to p75NGFR with antibodies. However, when NGF binding to p140trkA was reduced by the use of a synthetic peptide corresponding to amino acids 36–53 of human p140trkA, internalization of NGF was decreased. Thus, at least in PC12 cells, internalization appears to require binding of NGF to p140trkA, but occurs irrespective of p140trkA kinase activity and ligand occupancy of p75NGFR. The NGF triple mutant Lys‐32/Lys‐34/Glu‐35 to Ala, which has been demonstrated to bind to p140trkA, but not to p75NGFR, induced tyrosine phosphorylation more rapidly than wild‐type NGF. Likewise, NGF‐induced tyrosine phosphorylation was accelerated when NGF binding to p75NGFR was prevented with REX‐IgG. These findings indicate that NGF binding by p75NGFR may modulate NGF‐induced p140trkA kinase activity. © 1994 Wiley‐Liss, Inc.
Resolving three types of chloride channels in demyelinated Xenopus axons
Authors: Wu J. V. , Shrager P.
Published: January 2005

Axons from Xenopus sciatic nerve were demyelinated by intraneural injection of lysolecithin rendering the entire internodal axolema accessible to a patch electrode. In this region, three types of anion selective pores were found and characterized at the single‐channel level. These included outwardly rectifying, inwardly rectifying, and maxi Cl− channels. The outwardly rectifying Cl− channels (24 pS) are activated by depolarization with a weak voltage dependence of 42 mV per e‐fold change in open probability. The inwardly rectifying Cl− channels (27 pS) are insensitive to voltage, but can be blocked by internal application of 100 μM SITS or DIDS. The I‐V curves of rectifying channels are S‐shaped and can be fitted by a kinetic model with a single free energy barrier. The rectification may be related to the location of this barrier. The maxi Cl− channel (335 pS) is often open at the resting potential, but is inactivated by a large depolarization. The rectification, voltage dependence, and inactivation of these channels may contribute to the regulation of axonal Cl− balance and resting potential. © 1994 Wiley‐Liss, Inc.
Regulation of nitric oxide synthase activity in cortical slices by excitatory amino acids and calcium
Authors: Alagarsamy S. , Lonart G. , Johnson K. M.
Published: January 2005

Nitric oxide synthase (NOS) activity was determined in adult rat frontal cortex and hippocampus by measuring the conversion of L‐[3H]arginine to L‐[3H]citrulline. N‐methyl‐D‐aspartate (NMDA), but not kainate or α‐amino‐3‐hydroxy‐5‐methylisoxazole‐4‐propionate (AMPA), stimulated NOS activity. This effect was concentration dependent (EC50 ≈ 30μM) and was inhibited by tetrodotoxin, EGTA, Nω‐nitro‐L‐arginine (NOARG), Mg2+, phencyclidine, and (cis)‐4‐phosphonomethyl‐2‐piperidine carboxylate (CGS 19755), but not by 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione (CNQX). NOS activity was increased to an even greater extent by the calcium ionophores ionomycin and A23187 and by depolarization with 50 mM K+. Interestingly, neither caffeine nor 1‐aminocyclopentane‐1,3‐dicarboxylic acid (1S,3R‐ACPD), drugs that would be expected to increase intracellular Ca2+ concentration by release of Ca2+ from intracellular ryanodine‐ and inositol‐1,4,5‐trisphosphate‐sensitive stores, respectively, had any significant effect on NOS activity. It is concluded that NOS can be activated by NMDA binding to a classic NMDA glutamate receptor subtype as well as by depolarization or other agents that increase the influx of extracellular Ca2+. The paradoxical lack of effect of caffeine, as well as the inhibitory effect of tetrodotoxin, are discussed. © 1994 Wiley‐Liss, Inc.