bluuebunny/crossref_metadata_2025 huggingface dataset:
Modulation of neurofibromatosis type 1 gene expression during in vitro myoblast differentiation
Authors: Gutman D. H. , Cole J. L. , Collins F. S.
Published: January 2005

Neurofibromin, the protein product of the neurofibromatiosis type 1 (NF1) gene, has two alternate isoforms which are generated by alternative splicing of two exons. One of these isoforms containing exon 48a is expressed at highest levels in muscle. Since neurofibromin is a p21‐ras regulator and has been recently shown to be modulated during Schwann cell differentiation, we examined the expression of the NF1 gene product during in vitro muscle differentiation. Previous work demonstrated that C2 © 1994 Wiley‐Liss, Inc. C12murine myoblast cell differentiation could be blocked by the introduction of an activated p21‐ras protein. Using this model system, we demonstrate that differentiationg C2C12 cells upregulate the expression ofNF1 mRNA by 2 days of serum starvation concomitant with increased expression of nicotinic acetylcholine receptor mRNA. This upregulation of mRNA expression paralleled an increase in neurofibromin and N‐ras levels, but no change in the relative abundance of the isoforms containing exon 23a or exon 48a was observed during in vitro myoblast differentiation. The increase in neurofibromin levels paralleled a decrease in the levels of activated p21‐ras as assayed by in vivo 32Porthophosphate incorporation into p21‐ras. These results suggest that in vitro C2C12 cell differentiation is associated with a concomitant increase in NF1 gene expression and decrease in the proportion of activated p21‐ras. © 1994 Wiley‐Liss, Inc.
Cloning and expression of inducible nitric oxide synthase from rat astrocytes
Authors: Galea E. , Reis D. J. , Feinstein D. L.
Published: January 2005

In primary cultures of rat astroglial cells exposure to bacterial endotoxin lipopolysaccharide (LPS) causes induction of a Ca2+‐independent form of the nitric oxide synthase (iNOS) enzyme. We have now cloned the mRNA encoding astroglial iNOS using a combination of cDNA library screening and polymerase chain reaction (PCR) amplification with degenerate oligonucleotides directed against conserved regions of all NOS enzymes. The sequence of astroglial iNOS cDNA is highly similar to the mouse macrophage sequence, having an overall homology of 92% at the DNA level and 93% at the protein level. As in other NOSs, canonical binding sites for redox cofactors are present. The 3′‐untranslated region displays 4 consensus AU‐pentamers, 2 polyadenylation sites, and terminates in a stretch of 17 adenosine residues. In situ hybridization studies with LPS‐treated astrocyte cultures demonstrated the presence of iNOS mRNA in the majority of astroglial cells, identified by antibody staining to the glial fibrillary acidic protein (GFAP). PCR analysis showed that LPS stimulated synthesis of astrocyte iNOS mRNA, which was detected as early as 2 hr after exposure to LPS, peaked at 4 hr, and slowly declined over the next 20 hr. These results confirm that astrocytes can express iNOS and provide tools for the subsequent analysis of iNOS gene expression in rodent brain © 1994 Wiley‐Liss, Inc.
Peptide bond specificity of calpain: Proteolysis of human myelin basic protein
Authors: Banik N. L. , Chou C.‐H. , Deibler G. E. , Krutzch H. C. , Hogan E. L.
Published: January 2005

In order to determine the peptide bond specificity of calpain, human myelin basic protein (HMBP) was treated with purified calpain of bovine brain. Upon incubation, HMBP component I (HMBP‐1) was degraded into several peptides as demonstrated by sodium dodecy1 sulfate‐polyacrylamide gel electrophoresis. Component I was more susceptible to degradation than components II and III. HMBP degradation products were separated by high performance liquid chromatography (HPLC) and the cleavage sites in HMBP molecules were determined by peptide sequence analysis and by N‐ and C‐terminal analyses. The major cleavage site was found to be 94Val‐95 Thr with several minor cleavages at 49Arg‐50Gly, 18Ala‐19Ser, 23His‐24Ala, 27Gly‐28Phe, 59Asp‐60Ser, 70Gly‐71Ser, 97Arg‐98Thr, 110Ser‐111Leu, 145Asp‐146Ala, and 156Leu‐157Gly. These results indicate that calpain is involved in the limited proteolysis of human myelin basic protein and prolonged incubation causes further digestion of the large peptides. © 1994 Wiley‐Liss, Inc.
Distribution of plectin, an intermediate filament‐associated protein, in the adult rat central nervous system
Authors: Errante L. D. , Wiche G. , Shaw G.
Published: January 2005

Plectin is a high molecular weight protein originally identified and characterized as a major cytoskeletal component of the C6 rat glioma cell. Here we demonstrate by immunoblotting of crude intermediate filament (IF) protein preparations that plectin is a cytoskeleton‐associated component of the rat spinal cord. We then used avidin‐biotin peroxidase immunocytochemistry and indirect immunofluorescence to localize plectin within the adult rat central nervous system (CNS) and examine its distribution with respect to IF proteins. Plectin immunoreactivity is localized to all ependymal cells including the choroidal epithelial cells and tanycytes, Bergmann glial processes, radially oriented glial cells in the spinal cord, astrocytes in white matter, a subset of astrocytes in gray matter, a subset of motoneurons in the brainstem and spinal cord, and certain endothelial cells. Colocalization studies with neural If proteins show that plectin has a unique distribution pattern which most closely resembles, but is distinct from, that of vimentin. The few plectin positive neurons invariably also contain the neurofilament triplet proteins and peripherin, so that the ability of plectin to bind to the triplet proteins in vitro may reflect an in vivo interaction. The predominance of plectin at the inner ventricular boundaries of the nervous system as well as at the blood‐brain barrier is in line with the pattern of plectin expression in other tissues and suggests a general role for plectin in the maintenance of such junctional regions. © 1994 Wiley‐Liss, Inc.
Axotomy induces preprotachykinin gene expression in a subpopulation of dorsal root ganglion neurons
Authors: Noguchi K. , Dubner R. , De Leon M. , Senba E. , Ruda M. A.
Published: January 2005

The distribution of dorsal root ganglion (DRG) cell sizes that show changes in preprotachykinin (PPT) gene expression and substance P (SP) levels following axotomy was examined using RNA blot analysis, in situ hybridization histochemistry, and immunocytochemistry. PPT mRNA was induced in medium‐sized (1,000–2,000 μm2) and large‐sized (>2,000 μm2) cells in the DRG after axotomy. There was a 165% increase in the number of labeled cells after sciatic transection and a 260% increase after spinal nerve transection which results in axotomy of all the cells in the ganglion. The further increase after spinal nerve transection suggests that the induction occurred in axotomized neurons. PPT mRNA label was also present in a reduced number of small (<1,000 μm2) cells after axotomy. SP immunoreactivity was also induced in medium‐and large‐sized cells and reduced in small‐sized cells. Our findings suggest that the expression of the PPT gene and SP is differentially regulated in different subpopulations of DRG neurons after axotomy and is consistent with the hypothesis that tachykinins may be important in both sensory transmission and regeneration. © 1994 Wiley‐Liss, Inc.
Myelin basic protein content of aggregating rat brain cell cultures treated with cytokines and/or demyelinating antibody: Effects of macrophage enrichment
Authors: Loughlin A. J. , Woodroofe M. N. , Cuzner M. L. , Honegger P. , Comte V. , Matthieu J. ‐M.
Published: January 2005

The demyelinative potential of the cytokines interleukin‐1α (IL‐1α), interferon‐γ (IFN‐γ), and tumor necrosis factor‐α (TNF‐α) has been investigated in myelinating aggregate brain cell cultures. Treatment of myelinated cultures with these cytokines resulted in a reduction in myelin basic protein (MBP) content. This effect was additively increased by anti‐myelin/oligodendrocyte glycoprotein (α‐MOG) in the presence of complement. Qualitative immunocytochemistry demonstrated that peritoneal macrophages, added to the fetal telencephalon cell suspensions at the start of the culture period, successfully integrated into aggregate cultures. Supplementing the macrophage component of the cultures in this fashion resulted in increased accumulation of MBP. The effect of IFN‐γ on MBP content of cultures was not affected by the presence of macrophages in increased numbers. © 1994 Wiley‐Liss, Inc.
Human interleukin 1β: Corticotropin releasing factor and ACTH releasing and gene expression in the male rat: In vivo and in vitro studies
Authors: Parsadaniantz S. Mélik , Levin N. , Lenoir V. , Roberts J. L. , Kerdelhué B.
Published: January 2005

Numerous studies have shown that interleukin 1 (IL1), a cytokine secreted by macrophages, is capable of stimulating the hypothalamo‐pituitary‐adrenai (HPA) axis. Nevertheless, the sites involved in IL1 stimulation of the HPA axis remain, to data, subjects of controversy. In the present study, using in vivo and in vitro approaches, we tried to characterize the route by which IL1 acts on the HPA axis. In vivo, after an i.p. injection of human IL1β (1μg/rat), we measured plasma ACTH concentration, anterior pituitary (AP) ACTH content, hypothalamic (HT) corticotropin releasing factor (CRF) content, and also AP pro‐opiomelanocortin (POMC) and HT CRF gene expression. ACTH and CRF were measured by specific radioimmunoassays (RIAs), and solution hybridization nuclease protection assay was used for quantification of nuclear POMC precursor RNA and nuclear and cytoplasmic POMC and CRF mRNA. Human IL1β provoked an increase in ACTH plasma concentration, a decrease in AP ACTH content, and a prolonged increase in AP POMC primary transcript levels (around 100%). A significant increase in APPOMC primary transcript content was evident 30 min after injection of hIL1β, while cytoplasmic POMC mRNA levels were increased in the AP only at 4 hr after injection of hIL1β. We did not observe an effect of hIL1β on either HT CRF content or HT CRF cytoplasmic mRNA levels. In order to characterize a possible direct effect of hIL1β; at the Ap level, we used an AP perifusion system to analyse the effect of hIL1β; and CRH on ACTH release and on POMC gene expression. CRF (10−9M) applied as a 10‐minpulse provoked a marked rise in ACTH release and an 80% increase (P < 0.05) in POMC primary nuclear RNA transcript. Treatment of AP with hIL1β; (10−9M or 10−10) for 60 min did not affect ACTH release or POMC primary nuclear RNA transcript levels. The present study would suggest that the acute effect of hIL1β on ACTH secretion and synthesis is probably not due to a direct action of hIL1β at the AP level, but could result from a direct or indirect effect of IL1β on the release and synthese of hypothalamic CRF. Wiley‐Liss, Inc.
Understanding the CD4 molecule: Surface expression and function
Authors: Morriso W. J. , Offner H. , Vandenbark A. A.
Published: January 2005

Surface expression of the CD4 glycoprotein molecule is postulated to facilitate antigen recognition through the T cell receptor (TCR) and is itself a receptor for human immunodeficiency virus (HIV)‐gp120 glycoprotein. Both antigen‐stimulated TCR activation and HIV infectivity can be blocked by whole anti‐CD4 antibodies. Although selective modulation of CD4 from the surface by gangliosides (GM1) blocks HIV infectivity, it enhances associated TCR function. Enhanced TCR function has also been observed after intracellular delivery of synthetic CD4 mRNA‐antisense oligodeoxynucleotides (ODN) that block de novo synthesis of CD4. These specific CD4 modulations were mechanistically different from one another yet they both selectively removed the CD4 molecule from the T cell surface and enhanced antigen‐stimulated function through the TCR. The proposed role of CD4 during TCR function and HIV infectivity was developed, in part, according to decreases following CD4 antagonism by whole antibody or down‐modulation of CD4 by phorbol‐stimulated protein kinase C activity. Selective CD4 modulations have independently redefined the specific contributions of CD4 surface expression during T cell activation and may establish a role for CD4 receptor subtypes during HIV‐1 infenction of CD4+ cells. © 1994 Wiley‐Liss, Inc.
GO, a guanine nucleotide binding protein, is expressed during neurite extension in the embryonic mouse
Authors: Schmidt C. J. , Zubiaur M. , Valenzuela D. , Neer E. J. , Dräger U. C.
Published: January 2005

The developmental pattern of expression of the G protein αO subunit and GAP43 were compared by immunohistochemical staining of mouse embryos. Staining for αO and GAP43 was identical and detected throughout the developing nervous system, and the antigens first appeared in neurons at the beginning of neuronal differentiation. GAP43 and αO were not detected in regions containing only neuroblasts. These observations suggest that αO and GAP43 may not be required for the decision to pass from neuroblast to differentiated neuron, but may play a role in signal transduction during early neuronal development. © 1994 Wiley‐Liss, Inc.
Differentiation of oligodendrocytes cultured from developing rat brain is enhanced by exogenous GM3 ganglioside
Authors: Yim S. H. , Farrer R. G. , Hammer J. A. , Yavin E. , Quarles R. H.
Published: January 2005

Cultures consisting primarily of O‐2A progenitor cells and immature oligodendrocytes with a few microglia and astrocytes were obtained by shaking primary cultures from neonatal rat brain after 12‐‐14 days in vitro. Addition of 50 μg/ml exogenous Neu‐NAcα2‐3Galβ1‐1′ ceramide (GM3 ganglioside) to the cultures resulted in an increase in the number and thickness of cell processes that stained intensely for sulfatide and galactocerebroside (galC) in comparison to control cultures without added GM3. The treated cultures also contained fewer astrocytes than control cultures as revealed by immunostaining for glial fibrillary acidic protein (GFAP). Cells that immunostained for both GFAP and sulfatide/galC were very rare in control cultures but were frequently seen in the GM3‐treated cultures, suggesting that these may represent cells changing their direction of differentiation away from type II astrocytes toward oligodendrocytes under the influence of GM3. These effects on the developing rat oligodendrocytes were specific for GM3 ganglioside and were not produced by adding GM1, GM2, GD3, or GD1a to the cultures. Lactosyl ceramide and neuraminyl lactose were also ineffective. When control cultures were initially plated on polylysine and incubated with [14C]galactose, GD3 was the principal labeled ganglioside. However, as the control cells differentiated over time in culture without the addition of exogenous GM3 and produced increasing amounts of myelin‐related components, the incorporation of [14C]galactose into endogenous GM3 increased to become the predominant labeled ganglioside by 6 days after plating. Metabolic labeling of the GM3‐treated oligodendrocytes with [14C]galactose revealed increased incorporation into galC and sulfatide in comparison to control cultures, but a decreased labeling of endogenous GM3. Similarly, incorporation of an amino acid precursor into the myelin‐associated glycoprotein (MAG) was increased by GM3 treatment, but incorporation into myelin basic protein (MBP) was not affected. Although the overall effect of added GM3 was to decrease the phosphorylation of most proteins in the oligodendrocytes, including MBP, GM3 enhanced the phosphorylation of MAG. These findings indicate that GM3 ganglioside has an important role in the differentiation of cells of the O‐2A lineage toward myelin production, since differentiation is associated with increased metabolic labeling of endogenous GM3 in control cultures and is enhanced by the addition of exogenous GM3. © 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.
Serotonin inhibits outgrowth of goldfish retina and impairs the trophic effect of taurine
Authors: Lima L. , Matus P. , Urbina M.
Published: January 2005

The regeneration of explants prepared from goldfish retinas with a prior crush of the optic nerve is stimulated by the sulphur amino acid, taurine. Serotonin has been reported to modify survival, proliferation, and outgrowth of nervous tissue. In the present work we evaluated the effect of serotonin and some serotonergic agonists on the neuritic outgrowth from goldfish retinal explants. Serotonin, its precursor, 5‐hydroxytryptophan, and the 5HT1A receptor agonists, 8‐hydroxy‐2‐(di‐n‐propylamino)tetralin and busprione, inhibited the outgrowth. The blockers of serotonin uptake, imipramine and citalopram, were also inhibitors of neurite sprouting. Imipramine favoured the inhibitory effect of serotonin at 10 days in culture. The concentration of serotonin and its metabolite, 5‐hydroxyindoleacetic acid, decreased in the retina at 3 and 5 days after the crush of the optic nerve. Serotonin levels started to recover after 5 days post‐lesion, and the metabolite also increased. This indicates that the lesion increases the turnover rate of serotonin and this may be related to its role in regeneration. Serotonin concentration was elevated by the intraocular administration of its precursor, 5‐hydroxytryptophan, indicating that the capacity for synthesis was preserved after the crush, but that it was smaller in the post‐lesioned retinas. The trophic effect of taurine was impaired by a low concentration of serotonin, probably by opposing the final effect on growth via different targets. These results support a role of serotonin in the regeneration of goldfish retina probably through 5HT1A receptors. © 1994 Wiley‐Liss, Inc.
D2 dopamine receptor protein location: Golgi impregnation‐gold toned and ultrastructural analysis of the rat neostriatum
Authors: Fisher R. S. , Levine M. S. , Sibley D. R. , Ariano M. A.
Published: January 2005

The neostriatal distribution of D2 dopamine receptor protein has been assessed using subtype‐selective polyclonal antibodies generated against three unique polypeptide sequences of the receptor. The experimental tissues were processed by peroxidase based immunohistochemical procedures for routine light microscopy, Golgi impregnation‐gold toned morphological characterization, and correlative light/electron microscopy. The results demonstrated a regional gradient of D2‐like dopamine receptor expression in the neostriatum, where lateral portions in the nucleus exhibited more reactive cell bodies than medial portions. D2‐like expression was detected in the three populations of neostriatal neurons, i. e., the medium‐sized spiny projections neurons, and the medium‐ and large‐sized aspiny interneuron types. Morphometric measurements of labeled neurons verified that medium and large diameter neurons expressed the D2‐like receptor subtype. D2‐like immunoreactivity was distributed throughout the cytoplasm in dendritic processes, and in presynaptic terminal boutons. Immunoreactivity for the receptor protein was also detected in small, thinly myelinated axons, suggesting the possibilities of anterograde transport of the receptor from cell bodies in the substantia nigra to their neostriatal terminal fields, as well as from local axon collaterals of neostriatal projections neurons. These findings provide evidence of widespread distribution of the D2‐like receptor protein in neostriatal neurons, and showed that the presynaptic D2 receptors contain analogous epitopes to the postsynaptic receptor subtype. © 1994 Wiley‐Liss, Inc.
Expression of growth‐associated protein‐43 kD in Schwann cells is regulated by axon‐Schwann cell interactions and cAMP
Authors: Scherer S. S. , Xu Y.‐T. , Roling D. , Wrabetz L. , Feltri M. L. , Kamholz J.
Published: January 2005

We have examined the regulation of growth‐associated protein 43 kD (GAP‐43) in rat Schwann cells. In unlesioned adult nerves, GAP‐43‐immunoreactivity was restricted to non‐myelinating Schwann cells and unmyelinated axons. When adult nerves were transected to cause permanent axotomy, previously myelinating Schwann cells expressed progressively more GAP‐43‐immunoreactivity over 3 weeks and GAP‐43 mRNA levels increased over a similar time course. The peak level of GAP‐43 mRNA occurred at least 2 weeks later than that of nerve growth factor receptor, another marker of denervated Schwann cells. In contrast, after nerve‐crush, which allows axonal regeneration, many fewer Schwann cells had GAP‐43‐immunoreactivity, and the amount of GAP‐43 mRNA was markedly lower than in transected nerves. Forskolin, a drug that activates adenylate cyclase and mimics many effects of axon‐Schwann cell interactions, markedly reduced GAP‐43‐immunoreactivity and mRNA expression in cultured Schwann cells, whereas interleukin‐1 had no effect. These data demonstrate that axon‐Schwann cell interactions inhibit the expression of GAP‐43 in Schwann cells and that this effect is mimicked by forskolin. © 1994 Wiley‐Liss, Inc.
Decreased synaptic density in aged brains and its prevention by rearing under enriched environment as revealed by synaptophysin contents
Authors: Saito S. , Kobayashi S. , Ohashi Y. , Igarashi M. , Komiya Y. , Ando S.
Published: January 2005

Changes in synaptic density in various brain regions were assessed among different age groups of rats maintained in ordinary small cages, as determined by synaptophysin assay. The synaptophysin content in hippocampus decreases as early as in the adult stage. The most remarkable decrement occurs in occipital cortex. In other regions, synaptophysin contents decrease in senescence to 60–77% of the respective peak values during young and adult stages. The other rat group reared under enriched environment in a large cage until 30 months of age was examined for synaptic density, and was revealed to maintain the similar levels as in young, or even higher levels in frontal, temporal, entorhinal cortices and hippocampus. These results indicate that the synaptic density in cerebrum decreases in senescence and this decrease can be prevented by rearing under enriched environment. © 1994 Wiley‐Liss, Inc.
G‐protein γ7 subunit is selectively expressed in medium‐sized neurons and dendrites of the rat neostriatum
Authors: Watson J. B. , Coulter P. M. , Margulies J. E. , de Lecea L. , Danielson P. E. , Erlander M. G. , Sutcliffe J. G.
Published: January 2005

We used subtractive hybridization to isolate clones of γ7, a 68 residue G‐protein γ subunit. Northern blotting and in situ hybridization reveal that the γ7 subunit mRNA is expressed primarily in medium‐sized neurons of the neostriatum and nucleus accumbens and neurons of the olfactory tubercle, and at low levels in the dentate gyrus of the hippocampal formation and laminae II–III, and V of the neocortex. The γ7 mRNA is translocated into dendrites of neurons in the neostriatum and the dentate gyrus of the hippocampus. γ7 is expressed at relatively very low concentrations in peripheral tissues. The selective pattern of γ7 expression within the brain is highly reminiscent of those of the striatum‐enriched adenylyl cyclase ACST, dopamine receptors, and the α subunit of Golf, suggesting that, in striatum, γ7 may be a subunit of a Golfα‐containing G protein that couples dopamine receptors selectively to ACST.Copyright © 1994 Wiley‐Liss, Inc.
Motoneuron survival is not affected by the proximo‐distal level of axotomy but by the possibility of regenerating axons to gain access to the distal nerve stump
Authors: Tõrnqvist E. , Aldskogius H.
Published: January 2005

The aim of this study was to examine whether axotomy‐induced motoneuron death in adult mammals differ: (1) with the distance between the site of injury and the nerve cell body, and (2) if contact between the transected nerve stumps is established after the injury, compared with cases where contact is prevented.

The hypoglossal nerve of adult rats was transected either proximally in the neck (proximal injury) or close to the tongue (distal injury). The nerve stumps were then either deflected from each other in order to prevent axon regeneration into the distal nerve stump, or sutured. Three months later, the extent of nerve cell loss was examined bilaterally in cresyl violet‐stained sections of the hypoglossal nucleus. In addition, we examined hypoglossal neuron survival twelve months after a proximal nerve transection with prevented regeneration.

Our results show that there was no significant difference in neuronal survival after a proximal nerve transection compared with a distal one, neither if contact between the nerve stumps was established nor if it was prevented. However, contact between the transected nerve stumps increased the likelihood of neuronal survival significantly after both proximally and distally located injury compared to nerve injury with prevented regeneration. There was no significant decrease in nerve cell survival after twelve months with prevented reinnervation compared with survival after three months.

These observations indicate that the extent of axotomy‐induced motoneuron death in adult mammals does not correlate with the proximo‐distal level of peripheral injury. Furthermore, early contact with the distal stump and/or target musculature is a significant factor for the survival of axotomized motoneurns. However, more than 50% of the original nerve cell population survives a considerable time even after permanent disconnection from the target. Copyright © 1994 Wiley‐Liss, Inc.
Effects of the N‐acetylgalactosaminyltransferase inhibitor on cultured cerebral cells
Authors: Grabois V. R. , Conde C. B. , Caputto R.
Published: January 2005

The inhibitor preparation of the UDP‐N‐acetylgalactosamine: GM3, N‐acetylgalactosaminyltransferase (EC 2.4.1.92) (GalNAc‐T) produces effects on the neurons and the glial (astrocytes) cells of the cerebrum in culture. The effect in culture is evidenced by aneuritogenesis, deficiency in the GalNAc‐T activity, and decrease in the content of gangliosides, proteins, and lipids. In isolated glial cells the effect is evidenced by cytoplasm vesiculation and premature cessation of proliferation compared with control culture. The pattern of gangliosides in the inhibited culture shows a decrease in the amount of GD1a with respect to GD3; this is compatible with the notion that the effect is due to an inhibitor of the GM2 synthase. The inhibitor effects are reverted when it is eliminated after 24 or 48 hr in the culture medium. © 1994 Wiley‐Liss, Inc.
Enhanced aggregation and β structure of amyloid β peptide after coincubation with C1Q
Authors: Webster S. , O'Barr S. , Rogers J.
Published: January 2005

Several lines of evidence now suggest that aggregation of soluble amyloid β peptide (Aβ) into a cross β sheet configuration may be an important factor in mediating potential neurotoxicity of Aβ. Synthetic Aβ has been shown to self aggregate in vitro. Here, we demonstrate that coincubation of freshly solubilized Aβ with C1q, a complement component known to bind Aβ in vitro and to colocalize with Aβ in vivo, results in as much as a 7‐fold enhancement of Aβ aggregation, as well as a 2–4‐fold enhancement of β structure within aggregates. The addition of C1q to preformed Aβ aggregates also results in significantly increased resistance to aggregate resolubilization. © 1994 Wiley‐Liss, Inc.
Inhibitors of free radical formation fail to attenuate direct β‐amyloid25–35 peptide‐mediated neurotoxicity in rat hippocampal cultures
Authors: Lockhart B. P. , Benicourt C. , Junien J.‐L. , Privat A.
Published: January 2005

The direct neurotoxic action of the β‐amyloid protein, the major constituent of senile plaques, may represent the underlying cause of neuronal degeneration observed in Alzheimer's disease. The apoptotic‐mediated neuronal death induced by β‐amyloid appears to reside in its ability to form Ca>2+‐permeable pores in neuronal membranes resulting in an excessive influx of Ca2+ and the induction of neurotoxic cascades. It is possible that during β‐amyloid exposure a Ca2+‐mediated increase in free radical generation may exceed the defensive capacity of cells and thus lead to cell death. Consequently, in the present study we have investigated the effect of a panoply of antioxidants and inhibitors of free radical formation on the development of β‐amyloid neurotoxicity. Acute exposure of rat hippocampal neurons to “aged” β‐amyloid25–35 peptide (5–50 m̈M) induced a slow, concentration‐dependent apoptotic neurotoxicity (25–85%) during a 6 day exposure. Co‐incubation of cultures with β‐amyloid25–35 peptide (25 μM) and inhibitors of nitric oxide synthase and/or xanthine oxidase (NG‐monomethyl‐L‐arginine [1 mM], Nω‐nitro‐L‐arginine [1 mM], oxypurinol [100 m̈M], allopurinol [100 m̈M]), important mediators of nitric oxide, superoxide, and hydroxyl radical formation, did not attenuate β‐amyloid neurotoxicity. Similarly, a reduction in free radical generation by selective inhibition of phospholipase‐A2 cyclooxygenase, and lipoxygenase activities with quinacrine (0.5 m̈M), indomethacin (50 m̈M), and nor‐dihydroguaiaretic acid (0.5 m̈M), respectively, did not reduce the proclivity of β‐amyloid to induce cell death. Exposure of cultures to catalase (25 U/ml) and/or superoxide dismutase (10 U/ml) as well as the free radical scavengers vitamin E (100 m̈M), vitamin C (100 m̈M), glutathione (100 m̈M), L‐cysteine (100 m̈M), N‐acetyl‐cysteine (100 m̈M), deferoxamine (5 m̈M), or haemoglobin (35 m̈g/ml) failed to attenuate the neurotoxic action of β‐amyloid. On the other hand, pre‐treatment of cultures with subtoxic concentrations of β‐amyloid peptide significantly increased the vulnerability of neurons to H2O2 exposure and suggest that β‐amyloid peptide renders neurons more sensitive to free radical attack. However, a potential β‐amyloid‐mediated increase in free radical formation is not a proximate cause of the neurotoxic mechanism of β‐amyloid in vitro. © 1994 Wiley‐Liss, Inc.
Effects of exogenous ATP and related analogues on the proliferation rate of dissociated primary cultures of rat astrocytes
Authors: Ciccarelli R. , di Iorio P. , Ballerini P. , Ambrosini G. , Giuliani P. , Tiboni G. M. , Caciagli F.
Published: January 2005

The effects of ATP (5–500 μM) were evaluated on the proliferation rate of cultured astrocytes by measuring 3H‐thymidine incorporation and by flow cytometric analysis of the cell cycle. Determinations after 16 hours showed that ATP present in the culture medium for the whole period caused a dose‐dependent reduction of cell proliferation, while if the exposure to ATP was limited to the first 8 hours, the proliferation was increased (always in a dose‐dependent manner). A time course study of 3H‐thymidine incorporation showed that, in the presence of ATP, 3H‐thymidine was incorporated at a slower rate than in controls; the replacement of the culture medium with an ATP‐free fresh medium, at the 8th hour, was followed by a 3H‐thymidine incorporation occurring at such a fast rate to overshoot the control values. High performance liquid chromatography (HPLC) analysis, carried out to identify purine compounds present in the culture medium during cell exposure to ATP, indicated that more than 95% of the added ATP was metabolized within 1 hr. Conversely, an increase of purine metabolites was measured, this accumulation being greater at the highest concentrations of added ATP. The presence of high levels of extracellular ATP catabolites suggested that these compounds may act on the regulation of cell replication via the different purine receptors. This hypothesis was tested and confirmed by using agonists and antagonists selective for the P1 and the P2 sites. One hundred μM 2methylthio‐ATP (2MeSATP), a P2Y agonist metabolized as fast as ATP, reproduced effects very similar to the ATP‐induced ones. On the other hand, the nonhydrolisable ATP analogue, adenosine 5′‐(beta, gamma‐imido)‐triphosphate (AMP‐PNP) at 100 μM, induced a mitogenic effect as well as the A2 site stimulation. On the contrary, the activation of A1 receptors by 5 μM R‐phenyl‐isopropyladenosine (R‐PIA) inhibited astrocyte proliferation; moreover, 100 nM 8‐cyclopentyl‐1,3‐dipropylxanthine (DPCPX), an A1 site antagonist, reversed the ATP‐induced inhibition of cell proliferation. These results indicate that exogenous ATP, as a consequence of its rapid extracellular breakdown, exerts a dual influence on astrocyte proliferation by the involvement of both P1 and P2Y receptors. These findings might be relevant to such pathological conditions of the central nervous system (CNS), as seizures, hypoxia or ischemia, in which great amounts of purines released in the brain can influence a reactive astrocyte proliferative response to injury. © 1994 Wiley‐Liss, Inc.
Differential expression of vomeromodulin and odorant‐binding protein, putative pheromone and odorant transporters, in the developing rat nasal chemosensory mucosae
Authors: Krishna N. S. Rama , Getchell M. L. , Margolis F. L. , Getchell Thomas V.
Published: January 2005

Expression of the putative pheromone and odorant transporter, vomeromodulin, was characterized in developing rat nasal mucosae using in situ hybridization and immunocytochemistry. Initial expression of vomeromodulin mRNA and protein was detected at embryonic day (E)16 in the maxillary sinus component of the lateral nasal glands. The abundance of mRNA and protein in the lateral nasal glands increased with age and reached a peak at postnatal day (P)27. Also at P27, vomeromodulin mRNA and protein expression was initiated in vomeronasal glands and posterior glands of the nasal septum. Comparison of the developmental expression of odorant‐binding protein, another carrier protein synthesized in the lateral nasal glands, with that of vomeromodulin demonstrated major differences. In contrast to vomeromodulin, odorant‐binding protein was not detected until postnatal day 2 in the ventral component of the lateral nasal glands and anterior glands of the nasal septum. These results suggest that the expression of vomeromodulin and odorant‐binding protein is developmentally and differentially regulated and confirms the suggestion that vomeromodulin may function in olfactory and vomeronasal perireceptor processes as a transporter for pheromones and odorants. In addition, the embryonic expression of vomeromodulin suggests its involvement in olfactory perireceptor processes in utero. © 1995 Wiley‐Liss, Inc.
Peripheral myelin protein 22: Facts and hypotheses
Authors: Suter Ueli , Snipes G. J.
Published: January 2005

Mutations affecting the peripheral myelin protein 22 (PMP22) gene are associated with inherited motor and sensory neuropathies in mouse (Trembler and Trembler‐J) and human (Charcot‐Marie‐Tooth disease type 1A and Dejerine‐Sottas syndrome). Although genetic studies have established a critical role of PMP22 in the formation and/or maintenance of myelin in the peripheral nervous system, the biological function of PMP22 in myelin and in non‐myelin forming cells remains largely enigmatic. In this Mini Review, we will summarize the current knowledge about PMP22 and discuss its hypothetical function(s) in a broad context. © 1995 Wiley‐Liss, Inc.
Appearance of inducible nitric oxide synthase in the rat central nervous system after rabies virus infection and during experimental allergic encephalomyelitis but not after peripheral administration of endotoxin
Authors: Van Dam Anne‐Marie , Bauer J. , Man‐A‐Hing W. K. H. , Marquette C. , Tilders F. J. H. , Berkenbosch F.
Published: January 2005

The cellular localization of inducible (iNOS) and constitutive (cNOS) nitric oxide synthase was studied in rats by immunocytochemical techniques involving specific iNOS and cNOS directed antibodies and by NADPH‐diaphorase histochemistry. Paraformaldehyde‐fixed vibratome sections of brains and cryostat sections of peripheral lymph nodes were studied of rats treated with endotoxin (2.5 μg/kg or 2.5 mg/kg i.v.), rats infected with rabies virus, and rats exposed to experimental allergic encephalomyelitis (EAE). Endotoxin‐treated animals showed no appearance of immunoreactive iNOS (ir‐iNOS) cells in the brain with the exception of a few microglial cells near the median eminence and some meningeal macrophages. In the same animals however, iNOS‐immunoreactive cells were found in peripheral lymph nodes. Neurons that stain positive for cNOS and for NADPH‐diaphorase could be observed in brains of control as well as of endotoxin‐treated animals with a similar distribution and staining intensity. In contrast, animals that had been infected with rabies virus or subjected to EAE, showed the appearance of ir‐iNOS‐positive cells in several brain areas. These cells are located near blood vessels and lesion sites. The majority of these cells are GSA‐I‐B4 isolectin‐positive and therefore are likely to represent macrophages. Our data suggest that increased production of nitric oxide may play a role in the altered brain functions in rabies‐infected and EAE rats. On the contrary, increased nitric oxide production is probably not involved in the non‐specific symptoms of sickness induced by endotoxin. © 1995 Wiley‐Liss, Inc.
Proliferative capacity of oligodendrocytes in the demyelinating twitcher spinal cord
Authors: Taniike M. , Suzuki Kinuko
Published: January 2005

The proliferative capacity of oligodendrocytes was investigated in the spinal white matter of the twitcher mouse, a murine model of a genetic demyelinating disease globoid cell leukodystrophy (GLD), in which degeneration of oligodendrocytes due to metabolic perturbation has been well documented. In normal mice at 30 and 45 days of age, proliferating cells labeled with 5‐bromo‐2′‐deoxyuridine (BrdU) were scarce, and the majority of BrdU‐labeled cells did not immunostain with antibodies for oligodendrocytes, astrocytes, or microglia/macrophages. Only a few cells with markers for oligodendrocytes, carbonic anhydrase (CA), or the Pi form of glutathione‐S‐transferase (Pi), were labeled with BrdU. In the twitcher spinal cord, total numbers of BrdU‐labeled cells were almost 6 times that of the normal littermate mice at 30 days of age, and 28 times at 45 days of age. However, this increase was largely due to an increase of cells labeled with F4/80, a marker for the microglia/macrophages. CA or Pi positive cells only constituted less than 10% of all labeled cells. With progression of demyelination from 30—45 days, total numbers of CA positive or Pi positive oligodendrocytes decreased, but percentages of cells double‐labeled with BrdU and CA or Pi remained fairly constant. The results indicated that oligodendrocytes proliferated, to some extent, in the twitcher despite the genetic metabolic defect, and their decrease in number with progression of disease was not due to declined proliferation but rather cellular degeneration as the result of an intrinsic metabolic perturbation. © 1995 Wiley‐Liss, Inc.
PKA and PKC activation induces opposite glial fibrillary acidic protein (GFAP) expression and morphology changes in a glioblastoma multiform cell line of clonal origin
Authors: Arcuri Cataldo , Bocchini V. , Guerrieri P. , Fages C. , Tardy M.
Published: January 2005

Possible differentiation mechanisms were investigated in a glioblastoma multiform cell line (GL15) presenting an undifferentiated phenotype with weak glial fibrillary acidic protein (GFAP) and strong vimentin (VIM) expression. Serum‐free conditions induced time‐dependent increases of GFAP‐mRNA and GFAP protein levels, associated with a process‐bearing astrocytic morphology.

Activation of protein kinase C (PKC) by tumor promoter phorbol 12‐myrystate 13‐acetate (PMA) induced a rapid morphological differentiation and a decrease in GFAP mRNA, whereas the GFAP level remained unchanged. Such parameters were shown to characterize a physiological differentiation stage in astroglial cultures. Treatment of process‐bearing GL15 cells with dibutyryl cyclic AMP (dbcAMP), a protein kinase A (PKA) activator, induced a timedependent decrease in the GFAP mRNA and GFAP protein levels and reverted morphological changes induced by serum‐free conditions. Neither PMA nor dbcAMP influenced the VIM mRNA expression.

In GL15 cells, PKC and PKA activation have opposite effects. Understanding the role of these kinases in malignant transformation and in the in vitro differentiation process is of both basic and clinical interest. © 1995 Wiley‐Liss, Inc.
Cryogenic spinal cord injury induces astrocytic gene expression of insulin‐like growth factor I and insulin‐like growth factor binding protein 2 during myelin regeneration
Authors: Yao Da‐Lin , West N. R. , Bondy C. A. , Brenner M. , Hudson L. D. , Zhou J. , Collins G. H. , Webster H. deF.
Published: January 2005

To study injury‐induced astrocytic responses associated with regrowth of axons and regeneration of myelin, the method of Collins and colleagues was used to make focal cryogenic lesions in spinal cords of adult rats (Collins et al.: J Neuropathol Exp Neurol 45:742–757, 1986). The duration of cryogenic injury (CI), the size of the cryode, and its temperature were chosen to destroy all myelin sheaths and axons without producing cavities or hemorrhages. Messenger RNA and peptide distributions of insulin‐like growth factor I (IGF‐I), IGF‐I receptor (IGFR‐I), IGF binding protein 2 (IGFBP‐2), glial fibrillary acidic protein (GFAP), and myelin basic protein (MBP) were studied 3–56 days after CI by in situ hybridization and immunocytochemistry. At 3 days, vimentin‐positive, GFAP‐negative astrocyte‐Iike cells in the lesion expressed IGF‐I mRNA and peptide and 7 days after CI, both were expressed by typical GFAP‐positive, hypertrophic astrocytes, many of which also were vimentin‐positive. Levels of IGF‐I, IGFBP‐2, and GFAP mRNA and peptide were higher in lesion astrocytes after 14 days. They attained maximum levels at 21–28 days before declining to near control levels at 56 days. Decreasing relative levels of oligodendroglial MBP mRNA were found in and around lesions 7–14 days after CI; subsequently, rising levels accompanied remyelination. At 28 and 56 days after CI, some transferrin‐positive, oligodendroglia‐like cells also were immunostained by anti‐IGFR‐I. Our findings suggest that early astrocytic production of IGF‐I and IGFBP‐2 may be involved in the myelin regeneration which occurs in this model of spinal cord injury. © 1995 Wiley‐Liss, Inc.

This article is a US Government work and, as such, is in the public domain in the United States of America.
Heterogeneity of human neuroblastoma cell lines in their proliferative responses to basic FGF, NGF, and EGF: Correlation with expression of growth factors and growth factor receptors
Authors: Janet T. , Lüdecke G. , Otten U. , Unsicker Klaus
Published: January 2005

Growth factors can induce both proliferation or differentiation of neuroblastoma (NB) cells through interaction with specific receptors. Using two automated colorimetric assays for determinations of cell numbers, the present study demonstrates that (a) different NB and neuroepithelioma cell lines show distinct responses, both qualitatively and quantitatively, to basic FGF (bFGF), NGF, and EGF(b) even closely related NB cell lines (e.g., SK‐N‐SH, SH‐SY5Y, and SHEP) do not respond uniformly to these factors; c) responses of the two neuroepithelioma cell lines employed (SK‐N‐MC and CHP‐100) differ, but match those of certain NB cell lines; and d) two growth factors, bFGF and EGF, may both stimulate or inhibit proliferation, depending on the cell line studied. Specifically, IMR‐32, SK‐N‐SH, and SH‐SY5Y showed a mitogenic response to each growth factor. Maximal proliferative responses ranged from 204–355% as compared to controls (100%). GICAN was stimulated by NGF (199%), and SK‐N‐MC and NMB by EGF (282 and 140%, respectively), but other factors were ineffective. CHP‐100 and GIMEN were inhibited by bFGF. NGF and EGF were not effective on CHP‐100 cells, while EGF caused an arrest of mitogenic activity in GIMEN cells, and NGF stimulated their proliferation. Cell lines SHEP and LAN1 did not respond to any factor. To begin to analyze putative relationships of growth factor responsiveness and growth factor/growth factor receptor expressions, IMR‐32, GIMEN, and LAN1 cell lines were studied for the presence of bFGF, NGF, FGF receptors (R)‐1 (flg) and FGFR‐4, trk, and low‐affinity NGF receptor (p75) mRNAs. All three cell lines expressed bFGF and NGF mRNA, but not the FGFR‐1, FGFR‐4, trk, and p75 mRNAs. These results suggest extremely diverse patterns of NB/neuroepithelioma cell responsiveness to “mitogenic” growth factors and no overt correlation between such responses and growth factor/growth factor receptor expression. © 1995 Wiley‐Liss, Inc.
Effects of growth factors on phosphatidylinositol‐3 kinase in astroglial cells
Authors: Pomerance Martine , Gavaret J.‐M. , Breton M. , Pierre M.
Published: January 2005

Growth factors differently regulate astroglial cell differentiation and proliferation. In an effort to understand the early intracellular events promoted by growth factors in astroglial cells, we have determined the effects of insulin‐like growth factor I (IGF1), insulin, platelet‐derived growth factor (PDGF), epidermal growth factor (EGF) and fibroblast growth factors (FGFs) on phosphatidylinositol‐3 kinase (PI(3)‐kinase).

In astroglial cells cultured in serum‐free medium, IGF1, PDGF, and EGF, which stimulate cell proliferation, increased PI(3)‐kinase activity immunoprecipitated with anti‐phosphotyrosine antibodies as shown by thin layer chromatography and high performance liquid chromatography. FGFa and FGFb, which strongly stimulate proliferation, glutamine synthetase, and deiodinase activities and modify cell morphology, have no effect on PI(3)‐kinase activity. Addition of 1 nM PDGF, 10 nM IGF1, or 100 nM EGF to the culture medium rapidly stimulated PI(3)‐kinase activity which declined slowly after 2 min. The stimulation of PI(3)‐kinase increased with growth factor concentration. The maximum increase in PI(3)‐kinase activity occurred with 50 nM IGF1, 1 nM PDGF, or 100 nM EGF. Since insulin was active only at high concentration (1 μM), its effect was probably mediated through IGF1 receptors and not through IGF1 receptors and not through insulin receptors.

IGF1 and PDGF, to a lesser degree, also increased the PI(3)‐kinase activity associated with pp60c‐src protein. Immunoblots performed with an antibody directed against the p85‐subunit of the PI(3)‐kinase confirmed that IGF1 increased the number of PI(3)‐kinase molecules associated with phosphotyrosine‐containing proteins or with c‐src protein.

Each growth factor affects in a different manner the association of PI(3)‐kinase with phosphotyrosine‐containing proteins and with pp60c‐src and thus probably modulates intracellular signals downstream of PI(3)‐kinase in astroglial cells. © 1995 Wiley‐Liss, Inc.
Macrophage inflammatory protein 1‐α mRNA expression in an immortalized microglial cell line and cortical astrocyte cultures
Authors: Murphy Greer , Jia X.‐C. , Song Y. , Ong E. , Shrivastava R. , Bocchini V. , Lee Y. L. , Eng L. F.
Published: January 2005

Macrophage inflammatory protein 1 (MIP‐1) is a recently characterized inflammatory and chemokinetic cytokine. Proinflammatory stimuli have been shown to induce expression of MIP‐1 by macrophages. We hypothesized that microglia and astrocytes express MIP‐1α because of their many immunologic similarities to macrophages. MIP‐1α mRNA was examined with quantitative reverse transcription and polymerase chain reaction in an immortalized mouse microglial cell line (BV‐2) and in mouse cortical astrocyte cultures. We found that in both the BV‐2 microglial cell line and in astrocyte cultures, MIP‐1α mRNA was strongly induced by lipopolysaccharide and the phorbol ester PMA. MIP‐1α mRNA was reduced by dBcAMP, interferon‐γ, and PGE1. Dexamethasone decreased MIP‐1α mRNA levels in astrocyte cultures, but not in BV‐2 microglial cells. Interleukin‐1β, tumor necrosis factor α and MIP‐1α had no effect on MIP‐1α mRNA is expression. These findings demonstrate that MIP‐1α mRNA is expressed by cultured glial cells and is regulated by proinflammatory and anti‐inflammatory stimuli. MIP‐1α may be expressed by microglia and astrocytes in vivo, and may help modulate cerebral inflammation. © 1995 Wiley‐Liss, Inc.