bluuebunny/crossref_metadata_2025 huggingface dataset:
Protein kinase C in cultured adult human oligodendrocytes: A potential role for isoform α as a mediator of process outgrowth
Authors: Yong V. W. , Dooley N. P. , Noble P. G.
Published: January 2005

The extension of cellular processes from the oligodendrocyte soma is an early and critical event in myelin formation. Previous reports from this laboratory have implicated a role for protein kinase C (PKC) as an important intracellular mediator of this critical step in myelinogenesis. In the current study, the regrowth of fibers by adult human oligodendrocytes was examined and was found to be significantly enhanced by the PKC stimulator, 4β‐phorbol‐12,13‐didecanoate (PDB); this was accompanied by a 400–500% increase in oligodendroglial PKC activity. In contrast to other cell types, the increased PKC activity in oligodendrocytes was not followed by subsequent down‐regulation of the enzyme. The role of PKC in oligodendroglial process formation was further demonstrated by the ability of inhibitors of PKC to block the basal‐ or PDB‐enhanced fiber outgrowth. As well, studies employing isoform‐specific agonists implicated PKCα as the major determinant of fiber outgrowth by oligodendrocytes. The potential significance of PKC in myelin formation was further underscored by the observation that the synthesis of myelin basic protein, a prerequisite component for myelinogenesis, was increased by 2‐fold in PDB‐treated oligodendrocytes. Collectively, these observations suggest that PKC, in particular the α isoform, constitutes an important mediator in the initiation of myelin formation. © 1994 Wiley‐Liss, Inc.
Neurotrophin‐3 acquires NGF‐like activity after exchange to five NGF amino acid residues: Molecular analysis of the sites in NGF mediating the specific interaction with the NGF high affinity receptor
Authors: Kullander K. , Ebendal T.
Published: January 2005

Despite the large sequence similarity around 55–60% among the known NGF‐related neurotrophins, the members display different activities on different subset of neurons. Recent studies have shown that the various neurotrophins are ligands with high affinity to different receptors of the Trk family of tyrosine kinase receptors. We wanted to elucidate what specific parts of NGF replaced in neurotrophin‐3 (NT‐3) would result in NGF‐like receptor binding and biological activity. By studying evolutionarily conserved amino acid sequences not shared by NT‐3 and NGF and excluding parts which have been examined in earlier work with NGF and BDNF chimeras as well as taking advantage of the crystallographic data available for NGF, we decided to exchange three specific blocks of two or three amino acids in the human NT‐3 backbone for the corresponding residues in NGF. The NGF residues Asn‐Ile‐Asn (43–45), Val‐Phe (48,49) and Gln‐Ala‐Ala (96–98) were combined in pairs and are all shown to contribute NGF‐like activity in the context of NT‐3. The most efficient NGF‐like transformation was obtained by the exchange of Pro‐Val and Leu‐Val‐Gly in NT‐3 to the NGF residues Val‐Phe and Gln‐Ala‐Ala. This mutant reached 90% NGF activity, based on survival of sympathetic neurons, stimulation of fibre outgrowth from sympathetic ganglia, the ability to block high affinity NGF binding to PC12 cells and phosphorylation of gp140trk. Thus, the three mutants with paired combinations of the NGF residues as well as the NT‐3 housing all three blocks of NGF residues were able to mimic NGF activity. This activity is gained, although the mutated neurotrophin proteins do not lose the original NT‐3 activity as ascertained by the stimulation of neurite outgrowth from the Remak ganglion. The three mutated sites are situated in two β‐loops at one end of the NGF molecule, forming a cleft that could specifically interact with high affinity to the signalling NGF receptor gp140trk.Copyright © 1994 Wiley‐Liss, Inc.
Subnanomolar concentration of VIP induces the nuclear translocation of protein kinase C in neonatal rat cortical astrocytes
Authors: Oláh Z. , Lehel Cs. , Anderson W. B. , Brenneman D. E. , v. Agoston D.
Published: January 2005

At subnanomolar concentrations, vasoactive intestinal peptide (VIP) can act as an astroglial mitogen and as a secretagogue for neurotrophic substances released from glia (Brenneman et al.: J Neurosci Res 25:386–394, 1990). Here we report that treatment with subnanomolar (0.1 nM) VIP, that does not produce an increase in intracellular cAMP levels, induced the translocation of protein kinase C (PKC) from the cytoplasm to the nucleus in neonatal cortical astrocytes, as revealed by immunohistochemistry, Western blot analysis, and measurements of the enzyme activity. Western blot analysis of subcellular fractions, using PKC isotype‐specific antisera, showed PKC alpha as well as the two novel PKC isotypes, delta and zeta immunoreactivities, whereas PKC beta or gamma immunoreactivities were not detected. PKC alpha was associated predominantly with the cytosolic compartment, while PKC delta was found in the plasma membrane and in nuclear fractions. In contrast, PKC zeta was distributed ubiquitously within the major subcellular fractions. Treatment of the cells with 0.1 nM VIP caused a marked increase in nuclear PKC alpha and, to a lesser extent, PKC delta and PKC zeta immunoreactivities. Western blot analysis showed that a low (1 nM) concentration of phorbol, 12‐myristate, 13 acetate also caused the subcellular redistribution of PKC immunoreactivities from the cytoplasm to the nuclear fraction, similar to VIP treatment. Exposure of astrocytes to high concentrations (1 μM) of phorbol, 12‐myristate, 13 acetate resulted in the down‐regulation of PKC alpha and PKC delta, while distribution of PKC zeta immunoreactivities were only slightly altered. Measurements of Ca2+‐ and phospholipid‐dependent PKC activities also showed a VIP‐induced redistribution of PKC activity from the cytoplasmic to the nuclear fractions. These results suggest that PKC may be involved in the signal transduction process elicited by VIP binding to the high affinity VIP receptors present on cortical astrocytes. The observed changes in the nuclear localization of PKC alpha and PKC delta in response to subnanomolar VIP may play a role in mediating the cellular response(s) to this peptide during neurodevelopment. © 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.
Duration of expression of inducible nitric oxide synthase in glial cells
Authors: Park S. K. , Murphy S.
Published: January 2005

Lippolysaccharide (LPS) or a combination of interleukin (IL)‐1ß and interferon (IFN)‐γ cause transcriptional induction of a calcium‐independent nitric oxide synthase (NOS) in astrocytes and C6 glioma cells. LPS induction of NOS in C6 cells was evidenced by a small amount of nitrite accumulation as compared with cells exposed to IL‐1ß/IFN‐γ, but the maximal NOS activity achieved (as revealed by cGMP formation) was the same. The NOS activity induced by LPS in C6 cells was maximal at 4 to 8 hr and then rapidly decreased, while NOS activity induced by IL‐1ß/IFN‐γ slowly decreased after 4 hr. In addition, the effects of re‐presenting IL‐1ß/IFN‐γ to both astrocytes and C6 cells after maximal induction of activity of the inducible from of NOS were studied. The re‐addition of cytokines prolonged both NOS mRNA expression and also enzyme activity, suggesting effects at either the transcriptional (further induction) or translational level (mRNA stability). These results imply that the time course of NO production by induced astrocytes depends both upon the nature of the inducing stimulus and the frequency of the cells' exposure to it. © 1994 Wiley‐Liss, Inc.
Differential expression of α‐actin mRNA and immunoreactive protein in brain microvascular pericytes and smooth muscle cells
Authors: Boado R. J. , Pardridge W. M.
Published: January 2005

Hypertension has been linked to opening of the blood‐brain barier and may be related to the expression of the smooth muscle α‐actin gene in contractile cells at the brain microvasculature. However, the cellular origin (i.e., endothelial cells, pericytes, smooth muscle cells) of the α‐actin mRNA in the brain microvasculature is not clearly identified. Therefore, we investigated the abundance of actin mRNA by Northern blot analysis in isolated brain microvessels and in brain microvascular endothelia or pericytes in tissue culture. All samples showed the characteristic 2.1 kb transcript corresponding to cytoplasmic and δ isoform mRNA. The 1.7 kb transcript corresponding to smooth muscle α‐actin was detected in freshly isolated bovine brain microvessels, in primary cultures of brain microvasular pericytes, or endothelial cells; the latter cultures contain both endothelial cells and pericytes. The α‐actin mRNA was absent in a cloned bovine brain endothelial cell line. The relative abundance of the α/(+γ) actin transcript ratio was: cultured pericytes > freshly isolated microvessels > endothelial primary. The cellular distribution of the smooth muscle α‐actin immunoreactive protein was studied by immunocytochemistry in cytospun/methanol‐fixed isolated bovine brain microvessels with a monoclonal antibody directed to the amino‐terminal decapeptide of the smooth muscle α‐actin isoform. This antibody reacted strongly with precapillary arterioles of isolated microvessels, whereas no immunostaining was observed in either capillary endothelial cells or in pericytes. In conclusion, the αL‐actin mRNA is expressed in brain microvascular pericytes in tissue culture, but the immunoreactive α‐actin protein is not expressed in brain microvascular pericytes in vivo. These data suggest that either (1) α‐action gene expression is induced in capillary pericytes in tissue culture or (2) α‐action mRNA in brain capillary pericytes in vivo is subject to translational repression resulting in no detectable α‐actin protein under normal conditions. © 1994 Wiley‐Liss, Inc.
C‐terminal CTII motif of 2′,3′‐cyclic nucleotide 3′‐phosphodiesterase undergoes carboxylmethylation
Authors: Cox M. E. , Gao E. N. , Braun P. E.
Published: January 2005

Eukaryotic proteins with a carboxyl‐terminal CaaX motif are modified by isoprenylation and subsequently processed by proteolysis of the three terminal amino acids and carboxylmethylation of the exposed cysteine residue. The myelination‐associated 2′,3′‐Cyclic Nucleotide 3′‐Phosphodiesterase (CNP) has a C‐terminal CTII sequence and is isoprenylated; however, no examples of subsequent processing exist when threonine, a polar residue, is located adjacent to the cysteine. Here we show that CNP is capable of being carboxylmethylated in both insect cells and glioma cells. This processing is dependent upon isoprenylation of the cysteine and can be inhibited with the isoprenylated cysteine derivative, N‐acetyl‐S‐farnesyl‐L‐cysteine. Although the role of the methyl group at the C‐terminus of other isoprenylated proteins is not fully understood, modulation of signal transduction pathways is strongly indicated. This modification of CNP may similarly regulate cell biological processes in myelinogenesis. © 1994 Wiley‐Liss, Inc.
Time course of ciliary neurotrophic factor mRNA expression is coincident with the presence of protoplasmic astrocytes in traumatized rat striatum
Authors: Asada H. , Ip N. Y. , Pan L. , Razack N. , Parfitt M. M. , Plunkett Robert J.
Published: January 2005

Adrenal grafting for Parkinson's disease has led to modest functional improvement despite poor graft survival. One explanation is a neurotrophic response within the traumatized striatum. This study was undertaken to investigate the time course of the astrocytic response in vivo and in vitro, and the expression of ciliary neurotrophic factor (CNTF) mRNA following striatal injury. Unilateral stereotaxic biopsy of the rat striatum was performed and gelatin sponge (gelfoam) was immediately placed into the biopsy cavity. Rats were sacrificed on days 1,3,5,7,14, and 28 post biopsy. Immunohistochemical staining of the traumatized striatum with antibodies to glial fibrillary acidic protein (GFAP) was carried out. The reactive astrocytes which appeared within 7 days after trauma were mostly protoplasmic on the basis of morphology, and maximal on day 7, being 30 times the level in the normal striatum. After day 7, fibrous astrocytes appeared and increased up to day 28, while protoplasmic astrocytes decreased. In addition, immunocytochemical double staining of short term cultured astrocytes from the traumatized striatum with anti‐A2B5 and anti‐GFAP antibodies revealed that 84% and 90% of astrocytes were type 1 astrocytes on days 3 and 7, respectively; however, by day 28 47% of astrocytes were type 2. Northern blot analysis revealed that CNTF mRNA expression was up‐regulated and peaked on day 7, coincident with a predominance of protoplasmic astrocytes in vivo and type 1 astrocytes in vitro, respectively. These finding suggest that the expression of CNTF mRNA is part of the early astrocytic response to trauma, particularly associated with protoplasmic astrocytes in vivo and type 1 astrocytes in vitro. We conclude that reactive astrocytes are likely candidates to produce the neurite promoting activity seen in previous studies after trauma in the striatum. CNTF may represent an early signal in the astrocyte‐mediated neurotrophic and neurite promoting responses. © 1995 Wiley‐Liss, Inc.
Release of endogenous catecholamines from the striatum and bed nucleus of stria terminalis evoked by potassium and N‐methyl‐D‐aspartate: In vitro microdialysis studies
Authors: Aliaga E. , Bustos G. , Gysling Katia
Published: January 2005

Induced release of endogenous dopamine and noradrenaline from coronal slices containing the striatum and the bed nucleus of the stria terminalis, respectively, was studied by means of in vitro microdialysis. A Ca+2‐dependent and reserpine‐sensitive K+‐induced release of catecholamines was detected in both nuclei. We confirmed that N‐methyl D‐aspartate (2.5 and 5.0 mM in the dialysis perfusion solution) induces the release of dopamine from the striatum, and this effect was blocked by prior dialysis perfusion with 500 μM MK‐801, a noncompetitive N‐methyl‐D‐aspartate receptor antagonist. Infusion of N‐methyl‐D‐aspartate (1–10 mM) or glutamate through the dialysis probe did not produce any detectable modification in the extracellular levels of noradrenaline in the bed nucleus of the stria terminalis. In addition, perfusion with D‐serine (100 μM)alone or in the presence of desipramine (10 μM) resulted in a slight increase in extracellular noradrenaline in the bed nucleus of the stria terminalis. However, N‐methyl‐D‐aspartate in the presence of D‐serine and desipramine produced a marked increase in extracellular noradrenaline from the bed nucleus of the stria terminalis. These results indicate that N‐methyl‐D‐aspartate receptors might regulate the release of noradrenaline from the bed nucleus of the stria terminalis as is the case of dopamine release in the striatum. The in vitro microdialysis seems to be a suitable complement to the in vivo microdialysis for the study of catecholamine release in discrete regions of the central nervous system and its local regulation by excitatory amino acid receptors. © 1995 Wiley‐Liss, Inc.
Survival, development, and electrical activity of central nervous system myelinated axons exposed to tumor necrosis factor in vitro
Authors: Dugandžija‐Novaković S. , Shrager Peter
Published: January 2005

Spinal cord explants from CD‐1 mouse embryos were cultured in Maximow slide assemblies to promote myelin development. At about 20 days in vitro, recombinant human or mouse tumor necrosis factor alpha (TNFa) was added. Observed 3–8 days later, myelin was largely intact. The myelin blistering and oligodendrocyte damage seen in other strains were generally absent. Axonal conduction was measured optically through the use of a voltage‐sensitive dye. Action potential shape, conduction velocity, and refractory period were all unchanged by exposure to TNFa. Two series of explants were grown with TNFa present continuously throughout the culture period. Observed with light and electron microscopy, myelin developed in at least 50% of the explants treated with recombinant mouse TNFα and 80% of those exposed to recombinant human TNFα. Optically recorded action potentials were of normal shape and refractory period. Conduction velocities were slightly lower than controls. CD‐1 mouse central nervous system contains TNFα receptors and yet was resistant to myelin damage. The apparent strain specificity of TNFα disruption of myelin may result from more indirect modes of action, including interaction with other cytokines produced by glial cells. Survival of axonal conduction suggest that Na+ channel function remains intact following TNFα exposure. © 1995 Wiley‐Liss, Inc.
AMPA‐selective glutamate receptor subunits in the rat hippocampus during aging
Authors: Nicoletti V. G. , Condorelli D. F. , Dell'Albani P. , Ragusa N. , Stella Anna Maria Giuffrida
Published: January 2005

The levels of mRNAs for the subunits of α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazole propionate (AMPA)‐selective glutamate receptors (GluR‐1, ‐2, ‐3, ‐4) in the rat hippocampus during aging were measured by Northern blotting. The distribution of these receptors was also examined at the protein level by immunoblotting using antibodies to GluR‐1 and to an epitope common to GluR‐2 and GluR‐3 (denoted GluR‐2/3). During aging a significant decrease of GluR‐1 protein, but no change in the corresponding mRNA level, was observed. No differences in the level of GluR‐2/3 protein and GluR‐2, ‐3, and ‐4 mRNAs at the various ages examined (4, 12, and 24 months) were detected. Our results show that AMPA receptors are only slightly influenced by the aging process in the rat hippocampus. The slight decrease in GluR‐1 protein content, not accompanied by a parallel decrease in the GluR‐1 mRNA level, might be explained by a decreased translational efficiency or an increased protein degradation of the GluR‐1 subunit. © 1995 Wiley‐Liss, Inc.
Modulation of ischemic signal by antagonists of N‐methyl‐D‐aspartate, nitric oxide synthase, and platelet‐activating factor in gerbil hippocampus
Authors: Zablocka B. , Lukasiuk K. , Lazarewicz J. W. , Domańska‐Janik K.
Published: January 2005

Cerebral ischemia in the gerbil results in early hippocampal changes, which include transient activation and/or translocation of protein kinase C (PKC), increased enzymatic activity of ornithine decarboxylase (ODC), and elevated DNA binding ability of activator protein‐1 (AP1). The time‐course of all three of these postischemic responses was found to be almost parallel, peaking at 3 hr after the ischemic insult. The effectiveness of known modulators of postischemic morphological outcome (MK‐801, L‐NAME, and gingkolides BN 52020 and BN 52021) in counteracting the induction of PKC, ODC, and AP1 formation was tested. These drugs were administrated as followed: MK‐801 (a noncompetitive inhibitor of NMDA channel), 0.8 mg/kg i.p., 30 min before ischemia, and 5 min after the insult; L‐NAME (competitive inhibitor of NO synthase), 10 mg/kg i.p., 30 min before ischemia, and 5 mg/kg, 5 min after ischemia; BN52020 and BN52021 (inhibitors of platelet‐activating factor: PAF receptors) were administered as a suspension in 5% ethanol in water by oral route, 10 mg/kg for 3 days before ischemia. Three of these drugs, MK‐801, L‐NAME, and BN52021, significantly reduced ischemia‐elevated activity of PKC and ODC, whereas AP1 formation was only partially attenuated. Our observations implicate the existence of different mechanism(s) for postischemic PKC and ODC activation, which in turn is engaged in AP1 induction. © 1995 Wiley‐Liss, Inc.
Caldesmon and low Mr isoform of tropomyosin are localized in neuronal growth cones
Authors: Kira M. , Tanaka J. , Sobue Kenji
Published: January 2005

Neuronal growth cones move actively, accompanying changes in intracellular Ca2+ concentration. The movement of growth cones may partly depend on the actomyosin system, considering the presence of actin and myosin II. Yet, Ca2+‐sensitive regulatory proteins for the actomyosin system have not been identified in growth cones. In the present study, caldesmon, an inhibitory protein on actin‐myosin interaction, was detected in the growth cone fraction isolated from embryonic rat brain, using immunoblotting with the antibody to chicken gizzard caldesmon. Morphological evidence of caldesmon in growth cones of cultured rat neurons was obtained using the indirect immunofluorescence method. Since inhibition of caldesmon on actin‐myosin interaction can be overcome by calmodulin and Ca2+, caldesmon may be involved in the Ca2+‐dependent regulation in growth cone motility. Tropomyosin is another member of the actomyosin system whose function may be regulated by caldesmon in smooth and nonmuscle cells. A low Mr isoform of tropomyosin was distributed in the growth cone fraction. Using specific antibodies against tropomyosin isoforms, we further clarified morphologically that the low Mr isoform was localized in growth cones, but not the high Mr isoform. High Mr isoforms of tropomyosin were present in nonneuronal cells. Actin filaments in growth cones may be unstable, since low Mr tropomyosin binds to actin filaments with a lower affinity than high Mr isoforms. The instability of actin filaments may be suitable for the rapid movement and shape changes of growth cones. © 1995 Wiley‐Liss, Inc.
Hyaluronic acid through a new injectable nerve guide delivery system enhances peripheral nerve regeneration in the rat
Authors: Seckel Brooke R. , Jones D. , Hekimian K. J. , Wang K.‐K. , Chakalis D. P. , Costas P. D.
Published: January 2005

The use of non‐neural conduits to bridge gaps in peripheral nerves has been noted in the literature for many years. A logical extension of this concept is the introduction of neurotrophic or growth promoting factors into the lumen. We present here an injectable nerve guide that allows percutaneous access to the microenvironment of the regenerating peripheral nerve within the guide's lumen. Hyaluronic acid, a compound associated with decreased scarring and improved fibrin matrix formation, is added sequentially to the regenerating peripheral rat sciatic nerve via this injectable nerve guide. Assessment of nerve regeneration and reinnervation shows better conduction velocity, higher axon counts, and a trend toward earlier myelination with hyaluronic acid compared with saline. This work not only implies hyaluronic acid's role as an agent that aids nerve growth but also describes a new tool that allows percutaneous access to the milieu of a regenerating nerve. © 1995 Wiley‐Liss, Inc.
Effects of verapamil on the release of different neurotransmitters
Authors: Sitges María , Reyes A.
Published: January 2005

The effect of verapamil on resting and depolarization‐induced monoamine release was investigated in rat hippocampal synaptosomes prelabeled with [3H]‐5‐hydroxytryptamine (HT) or [3H]‐norepinephrine (NE) and rat striatal synaptosomes prelabeled with [3H]‐dopamine (DA). Verapamil (50 μM) completely abolishes high K+‐induced [3H]‐NE release, but paradoxically facilitates high K+‐induced [3H]‐5‐HT and [3H]‐DA release. All these high K+‐evoked responses were Ca2+ dependent. Verapamil does not modify [3H]‐NE baseline release, but increases dose dependently [3H]‐5‐HT and [3H]‐DA baseline release. Verapamil (10 μM, for 5 min) increases endogenous DA release (70%) and endogenous 5‐HT release (40%) independently on the presence of external Ca2+. The total amount of these monoamines (released plus retained by the preparation) and their metabolites (DOPAC and 5‐HIAA) was similar in control and verapamil‐treated synaptosomes. Verapamil displaces [3H]‐spiroperidol specific binding (Ki of 2.4 × 10−6M) and [3H]‐SCH‐23390 specific binding (Ki of 9 × 10−6M) from striatal synaptosomal membranes, and [3H]‐5‐HT specific binding (Ki of 3 × 10−5M) from hippocampal synaptosomal membranes. It is concluded that in addition to the Ca2+ antagonistic properties of verapamil on the Ca2+‐dependent, depolarization‐induced release of some neurotransmitters [gamma aminobutyric acid (GABA and NE)], another mechanism probably mediated by presynaptic receptors underlies the effects of verapamil on DA and 5‐HT release from discrete brain regions. © 1995 Wiley‐Liss, Inc.
Evidence that secretase cleavage of cell surface Alzheimer amyloid precursor occurs after normal endocytic internalization
Authors: Refolo L. M. , Sambamurti K. , Efthimiopoulos S. , Pappolla M. A. , Robakis N. K.
Published: January 2005

Three different treatments (methylamine, colchicine, and 18°C temperature block), known to disrupt normal endocytic internalization, significantly reduced the secretory cleavage of cell surface‐derived Alzheimer amyloid precursor (APP) in non‐transfected C6 cell cultures. Conversely, treatments with methylamine or colchicine had no significant effect on the secretion of total APP. Treatment of these cells with the lysosomotropic amine chloroquine resulted in a significant increase in the levels of both cell surface full‐length APP and cell surface‐derived secreted nexin II (NXII). Immunofluorescence analysis of C6 glioma cells transfected with APP751 indicated that under normal conditions, cell surface APP was internalized, and within 30 minutes was localized in discrete intracellular vesicles. These vesicles contained the endocytic tracer Texas red‐conjugated ovalbumin and probably represented late endosomes or lyso‐somes. However, treatment of the transfected C6 cultures with methylamine or colchicine prevented localization of cell surface APP in intracellular vesicles, suggesting that these treatments altered the normal intracellular trafficking of cell surface‐derived APP. Both the biochemical and immunofluorescence data are compatible with the suggestion that inhibition of normal endocytic internalization reduces the secretory cleavage of cell surface APP. Furthermore, our results suggest that following internalization, cell surface APP is cleaved by secretase(s) and secreted or routed to the lysosomes where it is degraded. © 1995 Wiley‐Liss, Inc.
Cloning of rat grp75, an hsp70‐family member, and its expression in normal and ischemic brain
Authors: Massa Stephen M. , Longo F. M. , Zuo J. , Wang S. , Chen J. , Sharp F. R.
Published: January 2005

Following metabolic stress a variety of gene products are induced in cells in the brain, some of which may protect the tissue from subsequent stresses. The heat shock proteins (hsps), in particular hsp70, have been widely studied in this context, but evidence for the involvement of known hsps in protection of the CNS is inconclusive. We have therefore undertaken the search for other stress‐induced proteins which may mitigate ischemic injury. Beginning with degenerate RT‐PCR, we have isolated a rat‐brain cDNA encoding a protein highly similar to human grp75, a mitochondrial member of the hsp 70‐family of stress proteins. It is also highly similar to two non‐mitochondrial proteins; mortalin, a senescence‐related gene product, and pbp74, a protein implicated in B‐cell peptide processing. Sequence structure and phylogenetic analyses predict mitochondrial localization and induction by a calcium ionophore and glucose deprivation in PC12 cells support its identification as rat grp75. In situ analysis of normal brain reveals an unusual distribution, with very high expression in neurons of the basal forebrain, reticular and subthalamic nuclei, globus pallidus, amygdala and elsewhere. grp75‐mRNA is upregulated following focal brain ischemia in a distinctive fashion. When the degree of injury is small, induction occurs in the area of injury, similar to the pattern observed for hsp70. However, when the injury is extensive, hsr is upregulated in neurons outside the ischemic area. The induction of grp75 may represent a sensitive marker of metabolically compromised tissue. © 1995 Wiley‐Liss, Inc.
Changes in neuronal mRNAs induced by a local inflammatory reaction
Authors: Lu X. , Richardson P. M.
Published: January 2005

Injection of Corynebacterium parvum into the rat dorsal root ganglion has previously been shown to cause an inflammatory reaction dominated by macrophages and to enhance regeneration of the central axons of primary sensory neurons. Here, neuronal mRNAs that are modified by nerve transection were analyzed by in situ hybridization following injection of C. parvum into the dorsal root ganglion. Neuronal concentrations of mRNAs for the growth‐associated protein (GAP‐43) and the immediate early gene c‐jun were increased by a local inflammatory response just as after axotomy. The concentration of mRNA for calcitonin gene‐related peptide (CGRP) was also increased in a constant subpopulation of sensory neurons after injection of C. parvum in contrast to its decrease following axotomy. The results are consistent with the hypothesis that some of the responses to sensory neurons to axotomy are sustained by macrophages which accumulate within the dorsal root ganglion after nerve injury. © 1995 Wiley‐Liss, Inc.
Bacterially expressed F1‐20/AP‐3 assembles clathrin into cages with a narrow size distribution: Implications for the regulation of quantal size during neurotransmission
Authors: Ye W. , Lafer E. M.
Published: January 2005

F1‐20/AP‐3 is a synapse‐specific phosphoprotein. In this study we characterize the ability of bacterially expressed F1‐20/AP‐3 to bind and assemble clathrin cages. We find that both of two bacterially expressed alternatively spliced isoforms of F1‐20/AP‐3 can bind and assemble clathrin as efficiently as preparations of F1‐20/AP‐3 from bovine brain. This establishes that the clathrin assembly activity found in F1‐20/AP‐3 preparations from brain extracts is indeed encoded by the cloned gene for F1‐20/AP‐3. It also demonstrates that posttranslation modification is not required for activation of the clathrin binding or assembly function of F1‐20/AP‐3. Ultrastructural analyses of the clathrin cages assembled by bacterially expressed F1‐20/AP‐3 reveals a strikingly narrow size distribution. This may be important for the regulation of quantal size during neurotransmission. We also express the 33 kD NH2‐terminus of F1‐20/AP‐3 in E. coli, and measure its ability to bind to clathrin triskelia, to bind to clathrin cages, and to assemble clathrin triskelia into clathrin cages. It has been suggested that the 33 kD NH2‐terminus of F1‐20/AP‐3 constitutes a clathrin binding domain. We find that the bacterially expressed 33 kD NH2‐terminus of Fl20/AP‐3 binds to clathrin triskelia, fails to bind to preassembled clathrin cages, and is not sufficient for clathrin assembly. The finding that the 33 kD NH2‐terminus of F1‐20/AP‐3 binds to clathrin triskelia but fails to assemble clathrin triskelia into clathrin cages is consistent with the published proteolysis studies. The finding that the 33 kD NH2‐terminus of F1‐20/AP‐3 fails to bind to clathrin cages is novel and potentially important. It is clear from these experiments that the 33 kD NH2‐terminus of F1‐20/AP‐3 is sufficient to carry out some aspects of clathrin binding; however it appears that defining the regions of the protein involved in clathrin binding and assembly may be more complex than originally anticipated. © 1995 Wiley‐Liss, Inc.
Abnormal expression and glycosylation of the large and small isoforms of myelin‐associated glycoprotein in dysmyelinating quaking mutants
Authors: Bartoszewicz Z. P. , Noronha A. B. , Fujita N. , Sato S. , Bö L. , Trapp B. D. , Quarles R. H.
Published: January 2005

The relative expression of large (L) and small (S) iso‐forms of the myelin‐associated glycoprotein (MAG) and their glycosylation were compared in developing spinal cord of quaking and control mice. Using anti‐sera specific for L‐ and S‐MAG, respectively, it was shown that S‐MAG is the principal isoform in quaking mice at all ages between 13 and 72 days, although L‐MAG was just detectable by western blotting at the early ages. Both L‐ and S‐MAG have higher apparent molecular weights in quaking mice than in controls. Experiments involving lectin binding and glycosidase treatment demonstrated that the higher molecular weight of MAG in the quaking mutant was due to a higher content of N‐acetyineuraminic acid residues linked α2‐3 to galactose as well as to more branching of oligosaccharide moieties indicated by a higher content of subterminal galactose residues. The total sialic acid measured by HPAE‐chromatography in purified quaking MAG was 40% higher than in control MAG. By contrast, quaking MAG contained less of the adhesion‐related, HNK‐1 carbohydrate epitope. Another difference was that a lower molecular weight form of MAG with predominantly high mannose oli‐gosaccharides was prominent in young quaking mice, but not in controls. The abnormalities of MAG expression related to splicing of its mRNA and glycosylation may contribute to the myelin pathology in quaking mutants. © 1995 Wiley‐Liss, Inc.
Oxystdrol (7β‐hydroxycholestesteryl‐3‐oleate) promotes serotonergic reinnervation in the lesioned rat spinal cord by reducing glial reaction
Authors: Giménez y Ribotta M. , Rajaofetra N. , Morin‐Richaud C. , Alonso G. , Bochelen D. , Sandillon F. , Legrand A. , Mersel M. , Privat A.
Published: January 2005

In the present study, following previous experience with electrolytic lesion of the rat brain, and subsequent reduction of reactive gliosis with 7b̃‐hydroxycholesterol derivatives (Bochelen et al.: Neuroscience 51:827‐834, 1992), we have performed a hemisection of the spinal cord in adult rats and investigated the influence of 7b̃‐hydroxycholesteryl‐3‐oleate (oxysterol) on the intensity of the astrocytic reaction and the axonal regeneration. We have shown here that local administration of liposomes containing this oxysterol reduced the intensity of the astroglial reaction on the sectioned side, as seen with immunocytochemical detection of glial fibrillary acidic protein (GFAP) and by in situ hybridization with a specific RNA probe. Moreover, radioautographic evaluation of astrocyte proliferation with tritiated thymidine evidenced a reduction of the astrocyte labelling index. In addition, double immunocytochemical detection of GFAP and polysialylated neural cell adhesion molecule (E‐NCAM) revealed a decrease of the expression of this molecule in reactive astrocytes of the treated animals. Finally, immunocytochemical detection of serotonin (5HT) was determined in the raphespinal projections, which constitute a major descending system. In treated animals, serotonergic axons originating from the intact side reinnervated the dorsal horn of the sectioned side, below the hemisection.

These results demonstrate that 7b̃‐hydroxycholesteryl‐3‐oleate can reduce the astrocytic reaction following spinal cord injury, promoting the serotonergic reinnervation of a denervated territory. © 1995 Wiley‐Liss, Inc.
A serum‐free, pyruvate‐free medium that supports neonatal neural/glial cultures
Authors: Martin F. C. , Wiley C. A.
Published: January 2005

Tissue culture media with serum generally cause excessive astrocyte proliferation in neonatal brain cultures, and often fail to support neonatal neurons. Published serum‐free media for brain cultures contain sodium pyruvate, which interferes with lactate dehydrogenase (LDH) assays for cell death. We wanted to use neonatal neural‐glial cultures in LDH assays while avoiding astrocyte proliferation, so we developed a serum‐free medium without sodium pyruvate. Our initial medium was based on that of Romijn et al. (J Neurosci Methods 23:75‐83, 1988), testing selected additives. Cell survival in 8‐10‐day‐old cultures was measured using 3‐[4,5‐Dimethylthiazol‐2‐yl]‐2,5‐diphenyltetrazolium bromide (MTT). N‐acetylcysteine, citrate, superoxide dismutase, ascorbate, supplemental amino acids, and high levels of transferrin improved survival. The optimized medium supported neonatal brain cells in reaggregates or in monolayers of 400 cells/mm2 for several weeks with large, healthyappearing neurons and very little astrocyte proliferation. Neurons stained strongly for the neuronal marker class III b̃‐tubulin and the synapse marker synaptophysin. Electron microscopy of reaggregate cultures demonstrated abundant neurons with synapses in a dense neuropil. This medium will be useful for various in vitro applications, especially those using LDH assays or requiring the use of neonatal cells. © 1995 Wiley‐Liss, Inc.
Expression of receptor protein tyrosine phosphatase‐σ (RPTP‐σ) in the nervous system of the developing and adult rat
Authors: Wang H. , Yan H. , Canoll P. D. , Silvennoinen O. , Schlessinger J. , Musacchio J. M.
Published: January 2005

The expression of receptor protein tyrosine phosphatase‐σ (RPTP‐σ) mRNA during rat development was examined by Northern blot and in situ hybridization analyses. Northern blot analysis revealed that the two transcripts (5.7 kb and 6.9 kb) had different spatial and temporal patterns of expression. The 6.9‐kb transcript was more abundant during embryonic development, whereas the 5.7‐kb transcript was more abundant during postnatal development and in the adult. In situ hybridization revealed that RPTP‐σ mRNA was widely expressed throughout the central and peripheral nervous system during embryonic development. Very high levels were seen in the ventricular zone, subventricular zone, cortex, dorsal root ganglia, cranial nerve ganglia, olfactory epithelium, and retina. During postnatal development the level of expression decreased in most brain regions. However, high levels continued to be seen in the hippocampus. Emulsion autoradiography revealed that the majority of RPTP‐σ mRNA is expressed in neurons. Northern analysis showed that cultured glial cells expressed the 6.9‐kb transcript, but not the 5.7‐kb. RPTP‐σ mRNA expression profiles were clearly distinct from those of leukocyte antigen‐related protein (LAR), a closely related RPTP. The spatiotemporal pattern of RPTP‐σ mRNA expression indicates that RPTP‐σ may play a role in the development of the nervous system. © 1995 Wiley‐Liss, Inc.
Glia‐to‐axon communication: Enrichment of glial proteins transferred to the squid giant axon
Authors: Sheller R. A. , Tytell M. , Smyers M. , Bittner G. D.
Published: January 2005

The transfer of newly synthesized proteins from the glial sheath into the axon is a well‐documented process for the squid giant axon. In this study, we used a novel approach to separate the transferred glial proteins (TGPs) from the endogenous axoplasmic proteins of the squid giant axon. Axoplasm, containing radiolabelled TGPs, was extruded as a cylinder and immersed in an intracellular buffer. After 1–30 min, the TGPs were enriched in the intracellular buffer, because they were eluted from the axoplasm into the intracellular buffer much faster than the endogenous axoplasmic proteins. Most of the TGPs enriched in the intracellular buffer did not pellet when centrifuged at 24,000 g for 20 min and were susceptible to protease digestion without the addition of Triton X‐100. Additionally, transmission electron microscopic autoradiography of intact axons, containing radiolabelled TGPs, suggested that most TGPs were not associated with vesicular organelles within the axon. We conclude that most of the TGPs are not contained within vesicles in the axoplasm of the squid giant axon, as would be expected if the mechanism of glia‐to‐axon transfer were conventional exocytosisendocytosis or microphagocytosis. © 1995 Wiley‐Liss, Inc.
Membrane properties of cultured glial cells obtained mechanically or by enzymatic dissociation: Differential alterations by gangliosides
Authors: Bouchon B. , Petegnief V. , Rebel G.
Published: January 2005

Rat astroglial cells were obtained either mechanically or by enzymatic dissociation and some properties of the primary and secondary cultures were studied. Differences in the ganglioside amount, taurine uptake, membrane fluorescence anisotropy, or their respective modulation by total brain gangliosides confirmed that although the two types of cultures are morphologically similar, they are biochemically distinct. © 1995 Wiley‐Liss, Inc.
Notochord and floor plate stimulate oligodendrocyte differentiation in cultures of the chick dorsal neural tube
Authors: Trousse F. , Giess M. C. , Soula C. , Ghandour S. , Duprat A.‐M. , Cochard P.
Published: January 2005

The regionalization of oligodendrocyte potentialities and the cellular interactions leading to the expression of the oligodendrocyte phenotype have been analyzed in the embryonic chick spinal cord. Dorsal and ventral regions of the spinal cord of 4‐day‐old embryos (E4) were cultivated separately. Oligodendrocyte differentiation was monitored at various times after explantation, using specific oligodendrocyte markers. After 2 weeks, several hundreds of differentiated oligodendrocytes were invariably observed in ventral cultures whereas significant numbers of oligodendrocytes failed to develop in dorsal spinal cord cultures. However, the E7 dorsal spinal cord was found to produce large numbers of oligodendrocytes, indicating that the ventral restriction of oligodendrocyte potentialities is transient. To test whether ventrally derived signals might influence oligodendrocyte differentiation, E4 dorsal spinal cord microexplants were cocultivated with notochord segments or with floor plate tissue. Numerous oligodendrocytes were found in dorsal explants associated with either tissue, notochord or floor plate, but not in dorsal explants cultivated alone, indicating that cells competent to be induced along the oligodendrocyte phenotype exist in the dorsal spinal cord. These results show that oligodendrocyte differentiation potentialities are initially restricted to the ventral spinal cord and suggest that ventrally derived signals from notochord and floor plate influence oligodendrocyte differentiation in the embryonic spinal cord. © 1995 Wiley‐Liss, Inc.
Chemotaxis and accumulation of nerve growth factor by microglia and macrophages
Authors: Gilad G. M. , Gilad V. H.
Published: January 2005

Astrocytes and microglia play a critical role in the reaction of the central nervous system (CNS) to trauma. Although both astrocytes and microglia can produce it, accumulation of immunoreactive nerve growth factor (the prototype neurotrophin important for the survival of several classes of neurons) was observed selectively in cultured microglia and macrophages, rather than in astrocytes. Furthermore, microglia were found to display chemotaxis toward a localized source of nerve growth factor and, as demonstrated by autoradiography, take up extracellular nerve growth factor. These findings suggest that microglia, the brain's own macrophages, participate in the regulation of nerve growth factor availability in a site‐specific manner. This novel function may assume a general importance both in the CNS and the peripheral nervous system at critical times after trauma when this neurotrophin is needed for nerve cell survival. © 1995 Wiley‐Liss, Inc.
Acetyl‐ and butyrylcholinesterase activities in the rat retina and retinal pigment epithelium
Authors: Sánchez‐Chávez G. , Vidal C. J. , Salceda R.
Published: January 2005

The acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) activities in the neural retina and retinal pigment epithelium (RPE) of adult rats were determined. The tissues were extracted with a saline buffer to release the soluble enzymes (S1) and the pellet re‐extracted with Triton X‐100 to detach the membrane‐bound enzymes (S2). Less than 5 % of the cholinesterase activity measured in retina and almost 30% of that assayed in RPE was due to BChE. About 20% and 10% of the AChE in retina and RPE was brought into solution with a saline buffer and the rest with a detergent‐containing buffer. Main AChE molecular forms of 10.5S (hydrophilic G4H), 9.5S (amphiphilic G4 A) and 3.OS (amphiphilic G1A) were identified in retina by subjecting the supernatant S1 to sedimentation analysis in sucrose gradients made with Brij 96. Amphiphilic G4 and G1 AChE were found in S2. Analysis of the soluble fractions obtained from RPE in the gradients made with Brij 96 revealed 16.OS (asymmetric A12), 10.5–10.0S (globular G4H + G4A), 4.5S (G2A), and 3.0S (G1A) AChE forms in S1, whereas G4A, G2A, and G1A enzyme molecules predominated in S2. Our results show that amphiphilic tetramers and monomers of AChE are abundant in neural retina, and enzyme tetramers, dimers, and monomers in RPE. The AChE in the neural retina might be involved in cholinergic actions. The enzyme function in the retinal pigment epithelium remains to be established. © 1995 Wiley‐Liss, Inc.
Differential regulation of bcl‐2, bax, c‐fos, junB, and krox‐24 expression in the cerebellum of Purkinje cell degeneration mutant mice
Authors: Gillardon F. , Bäurle J. , Wickert H. , Grüsser‐Cornehls U. , Zimmermann M.
Published: January 2005

Purkinje cell degeneration (pcd) is an autosomal recessive mutation in the mouse characterized by an almost complete loss of cerebellar Purkinje neurons between postnatal days 22 and 28. The pcd gene has not been identified, however, a relationship between activation of specitic genes and cell death has been suggested in other models of neuronal cell death. In the present study we analyzed the expression of several candidate cell death effector genes (bax, c fos, junB, krox‐24) and a cell death repressor gene (bcl‐2) in the cerebellum of pcd homozygotes and wild‐type mice. At postnatal day 22, when Purkinje cells start to degenerate, levels of c fos, junB, and krox‐24 mRNA increased about 5‐fold in mutants. To the contrary, the amount of bcl‐2 mRNA declined and bax transcripts remained unchanged compared to wild‐type animals. Immunoreactivity for c‐Fos and Jun could be detected exclusively in cerebellar Purkinje neurons of pcd mice but not in wild‐types, whereas the number of Bcl‐2 immunopositive Purkinje cells decreased significantly in mutants. Both double labeling experiments and immunostaining of consecutive sections revealed lack of colocalization of Jun with Bcl‐2. These results demonstrate an induction of members of the fos and jun family and a downregulation of antiapoptotic bcl‐2 in cerebellar Purkinje neurons that are destined to die. Fos and Jun transcription factor proteins may be implicated in the regulation of bcl‐2 expression and in the signal cascade leading to Purkinje cell death. © 1995 Wiley‐Liss, Inc.
Three isoforms of human myelin basic protein: Purification and structure
Authors: Deibler G. E. , Burlin T. V. , Stone A. L.
Published: January 2005

Myelin basic protein (MBP) occurs in multiple forms. Three of these isoforms from human MBP (HMBP) have been highly purified. HMBP, component 1 (18.5 kDa HMNP‐1), was purified by ion‐exchange chromatography at pH 10.6 in 2 M urea. During this ion‐exchange chromatography, a fraction (Fraction 3), which contained HMBP component 3 (monophosphorylated or deamidated 18.5 kDa) and 17.2 kDa HMBP, was collected and further purified by fast protein liquid chromatography, which separated 17.2 kDa HMBP and HMDP component 3. When the latter was subjected to limited thrombic digestion, all of HMBP component 3 not phosphorylated at theonine 98 was cleaved. This digestion mixture was separated on Sephadex, and yielded pure component 3, monophosphorylated at theonine 98 (HMBP 3pT98), for which phosphate analysis yielded approximately 1 mole P/mole protein, and NMR showed only one phosphorylation site present.

Circular dichroism (CD) studies were carried out on dilute solutions of HMBP‐1 (18.5 kDa), 17.2 kDa HMBP, and HMBP3pT98 (phosphorylated 18.5 kDa). The CD spectrum of HMBP‐1 was similar to that reported for rabbit MBP‐1 and bovine MBP‐1, but the spectra of 17.2 kDa HMBP and HMBP 3pT98 were distinctly different from HMBP‐1. When analyzed by best‐fit computations, 17.2 kDa HMBP showed about a 9% increase of ordered structure, and a greater increase, about 12%, was estimated for HMBP3pT98, attributable to β‐structure and β turn. © 1995 Wiley‐Liss, Inc.