bluuebunny/crossref_metadata_2025 huggingface dataset:
Molecular cloning and characterization of rat brain 2′, 3′ ‐cyclic nucleotide 3′ ‐phosphodiesterase isoform 2
Authors: Gravel M. , DeAngelis D. , Braun P. E.
Published: January 2005

We have isolated a cDNA coding for the larger isoform of the rat brain 2′,3′ ‐cyclic nucleotide 3′ ‐phosphodiesterase (CNP2), a protein associated with myelination in the central nervous system (CNS). The complete 420 amino acid sequence was deduced from the nucleotide sequence of the cDNA. Sequence comparisons show that rat CNP shares 96% homology with mouse, 84% with bovine, and 86% with human CNP. Errors in the published sequence of rat CNP1 have now been corrected. Comparisons with other proteins reveal several interesting conserved motifs, including two leucine repeat heptads, and two consensus motifs for phosphorylation in the N‐terminal domain of CNP2.© 1994 Wiley‐Liss, Inc.
Stimulation of reactive astrogliosis in vivo by extracellular adenosine diphosphate or an adenosine A2 receptor agonist
Authors: Hindley S. , Herman M. A. R. , Rathbone M. P.
Published: January 2005

Adenosine and its nucleotides adenosine triphosphate (ATP) and adenosine diphosphate (ADP) stimulate the proliferation of brain astrocytes in vitro and augment the effects of other growth factors. Following brain injury, hypoxia, or around solid tumors with necrotic centers, such as glioblastoma multiformes, high concentrations of adenine nucleotides and adenosine are released into the extracellular space; extracellular adenosine concentrations can rise 30–100‐fold to a concentration in excess of 100 μM. Increased concentrations of extracellular adenosine and adenine nucleotides may contribute to reactive astrocytic proliferation following brain injury. To test this hypothesis, adenosine, an adenosine analog 5′‐(N‐cyclopropyl)‐carboxamidoadenosine (CPCA), or ADP was microinjected into rat cortex. The number of glial fibrillary acidic prtein‐immunopositive cells was compared between the treated and contralateral saline‐injected hemispheres. Within 48 hr, astrocyte density around the CPCA (100 μM) infusion site was almost double that around the control saline infusion site. In hemispheres into which CPCA was infused, there was an increase in astrocytes in the subpial region along fiber tracts and around blood vessels, characteristic of Scherer's secondary structures found in association with malignant astrocytic brain tumors. The increased astrogliosis elicited by CPCA was abolished by coinfusion of the adenosine A2 receptor antagonist 1,3‐dipropyl‐7‐methylxanthine (DPMX). While microinjection of adenosine (1 mM) failed to stimulate astrogliosis, microinjection of ADP (500 μM) also resulted in a significant reactive astrogliosis and accumulation of astrocytes similar to Scherer's secondary structures. These data indicate that purine nucleosides and nucleotides may play a role in reactive astrogliosis following injury and may also play a role in stimulating the astrocyte migration responsible for Scherer's secondary structures around astrogliomas. © 1994 Wiley‐Liss, Inc.
Role for glial cells in regulating the functional expression of neuropeptide Y (NPY) neurons in aggregate cultures derived from dissociated fetal brain cells
Authors: Barnea A. , Cho G. , Lu G.
Published: January 2005

A series of studies from our laboratory have established an aggregate culture system of fetal rat brain cells expressing neuropeptide Y (NPY) which can serve as a model to study the role of glia‐neuron paracrine interactions in the developmental expression of NPY neurons. In this system, NPY production increases progressively with culture‐age and it is induced by forskolin (FOR) and phorbol 12‐myristate 13‐acetate (PMA). We addressed the following question: Is the functional expression of the NPY neurons impaired in the absence of glial cells (particularly astrocytes) and if so, can secretory products of aggregates composed of the full complement of brain cells ( intact aggregates) restore the function of the impaired NPY neurons? Aggregates were generated from 17‐day‐old fetal rat cortex and maintained in serum‐free medium for 13–15 days. Cytosine arabinoside (CA; doses of 0.5–8 μM) was added to the cultures on day 1 and the effectiveness in elimination of glial cells was verified on day 15 by measuring the incorporation of 3H thymidine into DNA and by immunostaining for the astrocyte marker glial fibrillary acidic protein (GFAP). Basal NPY production and FOR (10 μM) + PMA (20 nM) stimulated production of NPY on days 13–15 were taken as functional criteria. FOR + PMA induced ≈2‐fold increase in NPY production in control cultures (no CA). CA inhibited both basal and FOR + PMA induced production of NPY and DNA synthesis in a dose‐dependent manner: at 6 μM CA, basal NPY production was reduced by about 50%, FOR + PMA stimulated production of NPY and DNA synthesis were completely inhibited, and astrocytes were essentially eliminated. A competing dose of 100 μM deoxycytidine completely reversed the effects of 6 μM CA on DNA synthesis and basal and FOR + PMA stimulated production of NPY. Conditioned media derived from intact aggregates completely restored basal and partially (≈50%) restored FOR + PMA stimulated production of NPY in CA‐treated aggregates. These results implicate secretory products of glial cells (astrocytes?) and/or glial‐neuron paracrine interactions in regulating the developmental expression of NPY neurons in culture such that activation of the cAMP and protein kinase C pathways leads to increased production of NPY. © 1994 Wiley‐Liss, Inc.
Four structurally distinct neuron‐specific olfactomedin‐related glycoproteins produced by differential promoter utilization and alternative mRNA splicing from a single gene
Authors: Danielson P. E. , Forss‐Petter S. , Battenberg E. L. F. , deLecea L. , Bloom F. E. , Sutcliffe J. G.
Published: January 2005

Four structurally related neuron‐specific 1B426b mRNAs, designated AMY, BMY, AMZ, and BMZ, have been isolated from rat brain cDNA libraries. The four mRNAs are related to one another by their shared M region and by two pairs of alternative 5′ (A, B) or 3′ (Y, Z) regions. All four possible combinations were detected. The four transcripts are derived by differential promoter utilization (to generate A or B 5′ ends) and alternative splicing (to generate Y or Z 3′ ends) of the primary transcripts of the single D2Sutle gene. All four mRNAs were detected in most brain regions, but were enriched within the cortex and hippocampus. In the pituitary only the two A‐type and in the adrenal glands only the two B‐type mRNAs were detected. In situ hybridization shows a highly heterogeneous distribution across brain regions, paralleling the Northern blot results and additionally identifying the reactive cells as neurons. The cDNAs encode related glycoproteins of 125, 153, 457, and 485 amino acids, which have been detected immunochemically. The AMZ and BMZ proteins show significant sequence similarity with olfactomedin, an extracellular matrix protein of bullfrog olfactory epithelium, suggesting the possibility of a matrix‐related function for these rat glycoproteins in neurons and neurosecretory cells. © 1994 Wiley‐Liss, Inc.
Histamine‐mediated neuronal death in a rat model of Wernicke's encephalopathy
Authors: Langlais P. J. , Zhang S. X. , Weilersbacher G. , Hough L. B. , Barke K. E.
Published: January 2005

Three experiments were conducted to examine the role of histamine in neuronal degeneration in a rat model of Wernick's encephalopathy induced by an acute bout of pyrithiamine‐induced thiamine deficiency (PTD). In the first experiment, histamine levels in medial thalamus of freely moving PTD rats measured by microdialysis were increased (180% of controls) at a prelesion stage of thiamine deficiency (treatment day 12) and further elevated 48 hr later (380%) in the same animals when necrosis was evident. Histamine levels in dialysates of the hippocampus collected simultaneously from the same animals were unchanged at either stage of thiamine deficiency. Glutamate levels in microdialysates from the same animals were unchanged at the prelesion stage but were significantly elevated on the second collection day. In a second experiment, separate groups of PTD and pairfed control (CT) rats were infused continuously with either α‐fluoromethylhistidine (FMH; 80 mg/day, i. p.), an irreversible inhibitor of histamine synthesis, or saline. FMH pretreatment produced a significant protection against PTD‐induced neuronal loss within the midline‐intralaminar and anteromedial thalamic nuclei, but had no effect on damage to ventrolateral nuclei, anteroventral nucleus, or the mammillary bodies. In a third study, histamine (80 μg, free base) or vehicle was directly infused into the same region of medial thalamus dialyzed in experiments 1. Histamine infusion into prelesion PTD but not CT animals resulted in severe neuronal loss and gliosis. Infusion of vehicle into the same regions of PTD and CT rats produced a mild gliosis restricted to the needle tract with no evidence of neuronal loss. These observations together with recent evidence of a histamine enhancement of glutamate receptor activation suggest that early histamine release may contribute significantly to glutamate‐N‐methyl‐D‐aspartate (NMDA)‐mediated excitotoxic neuronal death in thiamine deficiency‐induced Wernicke's encephalopathy. © 1994 Wiley‐Liss, Inc.
Oligodendroglial cyclic AMP response element‐binding protein: A member of the CREB family of transcription factors
Authors: Sato‐Bigbee C. , Chan E. L. P. , Yu R. K.
Published: January 2005

Several laboratories have shown that cyclic AMP (cAMP) plays an important role in inducing oligodendrocyte differentiation and myelin synthesis. Our previous results have shown that oligodendrocytes contain a nuclear protein that binds to the DNA sequence TGACGTCA or cAMP response element (CRE) known to be involved in the transcriptional regulation of cAMP‐responsive genes. In this report the oligodendroglial CRE‐binding protein was further identified by using two different antibodies which specifically recognize the CRE‐binding protein known as CREB. In DNA‐shift assays CREB‐1 (X‐12) antibody interacted with the CRE‐protein complexes resulting in further retardation (“super shift”) of the mobility of the bands in the gels. Immunoprecipitation of oligodendroglial nuclear extracts with CREB(240) antibody prior to the DNA binding assays resulted in a lack of formation of CRE‐protein complexes. In addition immunoreaction with CREB(240) antibody identified the CRE‐binding species as a 45 kDa phosphoprotein. Immunocytochemical staining with CREB(240) antibody in oligodendrocytes from 10‐, 14‐, and 18‐day‐old and adult rats indicated that this protein in expressed before the appearance of myelin basic protein (MBP) which was used as a marker of myelin synthesis. Collectively, these observations support our previous results and indicate that the oligodendroglial CRE‐binding protein species is highly homologous to the CREB protein. The developmental expression of this CREB protein supports the idea of a possible role during the early stages of oligodendrocyte differentiation preceding the peak of myelin synthesis in rat CNS. © 1994 Wiley‐Liss, Inc.
Identification and characterization of batk, a predominantly brain‐specific non‐receptor protein tyrosine kinase related to Csk
Authors: Kuo S. S. , Moran P. , Gripp J. , Armanini M. , Phillips H. S. , Goddard A. , Caras I. W.
Published: January 2005

A novel cDNA, brain‐associated tyrosine kinase (Batk), was isolated from a rat hippocampal library and appears to encode a new member of the Csk subfamily of non‐receptor protein tyrosine kinases, with 52% overall amino acid identity to rat Csk. Batk resembles kinases of the Src family in that it contains a Src homology 2 (SH2) domain and an SH3 domain, followed by a tyrosine kinase domain. Analysis of incompletely spliced Batk cDNAs suggests that the genomic structure of Batk is similar to that of Csk with identical exon/intron boundaries. Batk also shows significant homology (86% overall amino acid identity) to the recently described human megakaryocyte‐specific Matk. Although Batk is 41 amino acids shorter than Matk, Southern blot analysis suggests that Batk might be a rat homolog of Matk. Batk is predominantly expressed in the brain, with lower expression in the spleen and undetectable expression in other tissues. In situ hybridization and Northern blot analysis show that Batk is widely distributed throughout the adult brain, being primarily expressed in neurons, including those of the hippocampus and cortex. In contrast, embryos appear to have markedly decreased expression levels. Analysis of postnatal day 1 brain suggests that Batk may be upregulated at birth throughout the brain except in the cerebellum. In view of its homology to Csk, a negative regulator of Src family tyrosine kinases, and its generalized expression in the adult brain, we suggest that Batk may function as a brain‐specific regulator of kinases involved in the development and maintenance of the nervous system. © 1994 Wiley‐Liss, Inc.
Transcription of the brain creatine kinase gene in glial cells is modulated by cyclic AMP‐dependent protein kinase
Authors: Kuzhikandathil E. V. , Molloy George R.
Published: January 2005

The brain creatine kinase (CKB) gene is expressed in a variety of tissues with highest expression seen in the brain. We have previously shown in primary rat brain cell cultures that CKB mRNA levels are high in oligodendrocytes and astrocytes and low in neurons (Molloy et al.: J Neurochem 59:1925–1932, 1992). In this report we show that treatment of human U87 glioblastoma cells with forskolin and IBMX, to elevate intracellular cAMP, induces expression of CKB mRNA from the transiently transfected rat CKB gene by 14‐fold and also increases expression from the endogenous human CKB gene. This induction of CKB mRNA (i) is due to increased transcription; (ii) occurs rapidly (with maximal induction after 6 hr; iii) requires the activity of protein kinase A (PKA), but (iv) does not require de novo protein synthesis and, in fact, is superinduced in the presence of cycloheximide. Given the role of oligodendrocytes in the energy‐demanding process of myelination and of astrocytes in ion transport, these results have physiological significance, since they suggest that changes in cellular energy requirements in the brain during events, such as glial cell differentiation and increased neuronal activity, may in part be met by a cAMP‐mediated modulation of CKB gene expression. Of particular importance is the possible modulation of CKB gene expression during myelinogenesis, since oligodendrocyte differentiation has been shown previously to be stimulated by increases in cAMP. Copyright © 1994 Wiley‐Liss, Inc.
Myelin basic protein mediates extracellular signals that regulate microtubule stability in oligodendrocyte membrane sheets
Authors: Dyer C. A. , Philibotte T. M. , Wolf M. K. , Billings‐Gagliardi S.
Published: January 2005

Treatment of cultured oligodendrocytes with a monoclonal antibody to galactocerebroside (GalC) triggers a cascade of events including the redistribution of membrane surface GalC over internal domains of MBP and loss of microtubular structures within the sheets (Dyer and Benjamins: J Neurosci 8:4307–4318, 1988; Dyer and Benjamins: J Neurosci Res 24:212–221, 1989). In this report, wild type and myelin basic protein (MBP)‐deficient shiverer oligodendrocytes were used to study the possible relationships between these events, and specifically to determine if MBP mediates signals which destabilize microtubular assemblies in cultured oligodendrocytes. We now show that MBP and GalC, which are both initially Triton X‐100 soluble, become Triton X‐100 insoluble following anti‐GalC binding and anti‐GalC:GalC complex redistribution, suggesting that the surface anti‐GalC: GalC complexes become associated with cytoplasmic MBP. Mediation of the signaling event by MBP is further demonstrated by (1) a decreased phosphorylation of MBP in wild type oligodendrocytes after antibody binding, and (2) the absence of responses, such as GalC redistribution and microtubule loss, in MBP‐deficient shiverer oligodendrocytes treated with anti‐GalC. Continuous activation of the GalC/MBP pathway for 7 days in wild type oligodendrocytes results in enlarged cell bodies and production of numerous microprocesses, a morphology that is similar to MBP‐deficient shiverer oligodendrocytes. A second signaling pathway which produces an opposite effect, i.e., the stabilization and apparent up‐regulation of microtubular structures in cultured oligodendrocyte membrane sheets, remains functional in shiverer oligodendrocytes. Thus, MBP appears to be important for mediating extracellular signals that cause a loss of microtubular structures in oligodendrocyte membrane sheets and abnormal morphology. Copyright © 1994 Wiley‐Liss, Inc.
The insulin‐like growth factor I system in the rat cerebellum: Developmental regulation and role in neuronal survival and differentiation
Authors: Torres‐Aleman I. , Pons S. , Arévalo M. A.
Published: January 2005

The developmental regulation of insulin‐like growth factor I (IGF‐I), its receptor, and its binding proteins (IGFBPs) was studied in the rat cerebellum. All the components of the IGF‐I system were detectable in the cerebellum at least by embryonic day 19. Levels of IGF‐I receptor and its mRNA were highest at perinatal ages and steadily decrease thereafter, although a partial recovery in IGF‐I receptor mRNA was found in adults. Levels of IGF‐I and its mRNA also peaked at early ages, although immunoreactive IGF‐I showed a second peak during adulthood. Finally, levels of IGFBPs were also highest at early postnatal ages and abruptly decreased thereafter to reach lower adult levels. Since highest levels of the different components of the IGF‐I system were found at periods of active cellular growth and differentiation we also examined possible trophic effects of IGF‐I on developing cerebellar cells in vitro. We found a dose‐dependent effect of IGF‐I on neuron survival together with a specific increase of the two main neurotransmitters used by cerebellar neurons, GABA and glutamate. Analysis of cerebellar cultures by combined in vitro autoradiography and immunocytochemistry with cell‐specific markers indicated that both Purkinje cells (calbindin‐positive) and other neurons (neurofilament‐positive) contain IGF‐I binding sites. These results extend previous observations on a developmental regulation of the IGF‐I system in the cerebellum and reinforce the notion of a physiologically relevant trophic role of IGF‐I in cerebellar development. © 1994 Wiley‐Liss, Inc.
Axonal transport of type III intermediate filament protein peripherin in intact and regenerating motor axons of the rat sciatic nerve
Authors: Chandan S. , Le Gall J.‐Y. , di Giamberardino L. , Filliatreau G.
Published: January 2005

Slow axonal transport of peripherin has been studied in the motor axons of both intact and regenerating rat sciatic nerves 7 days post‐crush. The studies were done by two‐dimensional gel electrophoresis after intraspinal injection of35S‐methionine.

In the first experiment, the sciatic nerves were removed 3 weeks after the radiolabeling pulse and cut into 6 mm segments. Each nerve segment was submitted to two‐dimensional gel electrophoresis and analyzed by an original procedure which allowed us to study the distribution along the nerve of the radioactivity associated with several proteins of the cytoskeleton, especially the intermediate filament proteins, peripherin, and the low molecular mass neurofilament protein, NF‐L. Peripherin was transported at two main rates: 66% of the total radiolabeled peripherin moved at 1.42 mm/day and the remainder moved at 2.28 mm/day. The radioactivity associated with NF‐L exhibited a similar pattern.

In the second experiment, similar intraspinal injections were made 7 days after a unilateral crush of the sciatic nerve. Regenerating nerves exhibited a clear SCa wave. However, in contrast to the intact nerves, the SCb wave could not be precisely defined in the regenerating nerves. Thus, the changes in the amount of transported proteins were analyzed in the SCa wave only. Autoradiograms of 2D‐PAGE revealed that in the regenerating axons, the quantity of transported peripherin in SCa was increased by 3.5‐fold. In contrast, the quantity of transported NF‐L was decreased by 1.6‐fold. The regenerating motor axons conveyed significantly greater (approximately twofold) amounts of labeled tubulins and actin than did intact motor axons.

Our results suggest that peripherin, although mainly conveyed by SCa, plays a role during the elongation process in addition to actin and tubulin. © 1994 Wiley‐Liss, Inc.
Long‐lasting accumulation of hemopexin in permanently transected peripheral nerves and its down‐regulation during regeneration
Authors: Madore N. , Sagot Y. , Guinaudy M. J. , Cochard P. , Swerts J. P.
Published: January 2005

We have previously demonstrated that hemopexin is present in the intact sciatic nerve and is overproduced in the distal stump after nerve transection (Swerts et al.: J Biol Chem 267:10596–10600, 1992). To get further insight into the function of this hemoprotein in nervous tissue, we have documented long‐term changes in hemopexin levels in permanently degenerated (transected) and regenerting (crush‐lesioned) sciatic nerves of adult rats, using immunochemical techniques. As early as a couple of days after nerve transection, the amount of hemopexin was raised in the distal stump and at the end of the proximal stump. Similarly, after a crush lesion hemopexin was rapidly increased at the injury site and in the distal part of the nerve. Subsequently, in transected nerves the level of hemopexin rose steadily and remained elevated, representing, three months after injury, over 20 times the amount found in intact contralateral nerves. In contrast, in crush‐lesioned nerves, hemopexin level declined progressively in a proximodistal direction and returned to basal values 2 months after injury, together with axonal regeneration. This long‐term increase in hemopexin in permanently degenerated nerves and its progressive return to normal levels during nerve regeneration suggests that hemopexin content could be regulated negatively, directly or indirectly, by growing axons. In turn, these results support the idea that hemopexin could be involved in the process of Wallerian degeneration and/or in nerve repair. © 1994 Wiley‐Liss, Inc.
Adenovirus‐mediated gene therapy of experimental gliomas
Authors: Perez‐Cruet M. J. , Trask T. W. , Chen S‐H. , Goodman J. C. , Woo S. L. C. , Grossman R. G. , Shine H. D.
Published: January 2005

The efficacy of adenovirus (ADV)‐mediated gene therapy to treat brain tumors was tested in a syngeneic glioma model. Tumor cells were transduced in situ with a replication‐defective ADV carrying the herpes simplex virus thymidine kinase (HSV‐tk) gene controlled by the Rous sarcoma virus promoter. Expression of the HSV‐tk gene enables the transduced cell to convert the drug ganciclovir to a form that is cytotoxic to dividing cells. Tumors were generated in Fischer 344 rats by stereotaxic implantation of 9L gliosarcoma cells into the caudate nucleus. Eight days later, the tumors were injected either with the ADV carrying the HSV‐tk (ADV‐tk) gene or a control ADV vector containing the β‐galactosidase (ADV‐βgal) gene and the rats were treated with either ganciclovir or saline. Tumor size was measured 20 days after implantation of 9L cells or at death. Rats treated with ADV‐βgal and ganciclovir or with ADV‐tk and saline had large tumors. No tumors were detected in animals treated with ADV‐tk and with ganciclovir at doses ≥80mg/kg. An infiltrate of macrophages and lymphocytes at the injection site in animals treated with ADV‐tk and ganciclovir indicated an active local immune reaction. In survival studies, all animals treated with ADV‐tk and ganciclovir have remained alive longer than 80 and up to 120 days after tumor induction whereas all untreated animals died by 22 days. These results demonstrate that ADV‐mediated transfer of HSV‐tk to glioma cells in vivo confers sensitivity to ganciclovir, and represents a potential method of treatment of brain tumors. © 1994 Wiley‐Liss, Inc.
Cytochrome P450 in rat astrocytes in vivo and in vitro: Intracellular localization and induction by phenytoin
Authors: Kempermann G. , Knoth R. , Gebicke‐Haerter P. J. , Stolz B.‐J. , Volk B.
Published: January 2005

Cytochrome P450IIB1,2 (nomenclature according to Nelson et al., DNA Cell Biol 12:1–51, 1993 and Volk et al., Neuroscience 42:215–235, 1991) immunoreactivity (P450‐IR) is associated with astrocytes both in vivo and in vitro. Although they are unevenly distributed throughout the brain with a preference for phylogenetically elder parts, no significant differences between astrocytes prepared from different brain regions were observed in astrocyte cultures. The percentage of strongly immunoreactive astrocytes decreased from 40% after 7 days in culture to 15% after 21 days. Essentially all astrocytes have a low but significant P450‐IR within this interval. Preembedding immunoelectron microscopy revealed peroxidase reaction products on the endoplasmic reticulum and on the outer membranes of mitochondrial and nuclear envelopes. Phenytoin (1 μM) added to the medium for 7 days significantly (1.22‐fold) increased the amount of total P450 in astrocyte homogenates as measured by spectrophotometry. Considerably more immunoreactive cells (1.5‐fold) were found in treated cultures than in controls. © 1994 Wiley‐Liss, Inc.
Lindane cytotoxicity in cultured neocortical neurons is ameliorated by GABA and flunitrazepam
Authors: Pomés A. , Frandsen A. A. , Suñol C. , Sanfeliu C. , Rodríguez‐Farré E. , Schousboe A.
Published: January 2005

The effect of lindane (γ‐hexachlorocyclohexane) on [35S]t‐butylbicyclophosphorothionate ([35S]TBPS) binding and GABA‐stimulated 36Cl− influx was investigated in cultured cerebral cortical neurons. In addition, the cytotoxic action of lindane as well as a protection by GABA and flunitrazepam were studied together with the ability of lindane to increase the intracellular concentration of free Ca2+. Lindane was found to be toxic to the neurons, an effect that could be completely prevented by the simultaneous presence of GABA (0.1 μM) and flunitrazepam (100 μM) and reduced by GABA alone. An interaction with the GABA receptor‐gated chloride channel was demonstrated by an inhibitory action of lindane on [35S]TBPS binding (IC50 188 ± 51 nM) and on GABA‐stimulated 36Cl− influx in the neurons. Lindane only marginally increased the intracellular Ca2+ concentration in the neurons. It is concluded that the cytotoxic action of lindane is mediated through interaction with GABA receptors in a manner essentially independent of changes in intracellular Ca2+ homeostasis. © 1994 Wiley‐Liss, Inc.
Tissue‐specific DNA methylation patterns of the rat glial fibrillary acidic protein gene
Authors: Condorelli D. F. , Nicoletti V. G. , Barresi V. , Caruso A. , Conticello S. , de Vellis J. , Stella A. M. Giuffrida
Published: January 2005

The glial fibrillary acidic protein (GFAP) is an intermediate filament protein, specific of the cytoskeleton of astrocytes in the central nervous system. In the present work, as a preliminary step to the study of glial‐specific gene expression, we cloned the rat GFAP gene, and we report the sequence of 1.9 kb of the 5′ flanking region, exon 1, and the majority of the first intron. By digestion with methylation‐sensitive restriction enzymes followed by Southern blot analysis, the methylation status of various CpG sites was examined in this genomic segment. We tested whether structural modification of the GFAP gene, such as DNA methylation, could be related to its tissue‐specific transcriptional activity. Therefore, we compared a GFAP‐expressing cell population (primary culture of astroglial cells), a mixed population of GFAP‐expressing and ‐nonexpressing cells (adult rat cerebral hemispheres), and a GFAP‐nonexpressing tissue (liver). In the 5′ flanking region we identified a CpG site at position −1176 whose level of methylation is inversely correlated to GFAP expression. In primary cultured astrocytes, 75% of the GFAP gene alleles were demethylated at this site, while the corresponding value obtained for the cerebral hemispheres was 45%, and for liver only 9%. On the basis of the sequence data, a CpG‐rich region (putative CpG island) was identified extending from −38 to + 347 and overlapping 80% of the first exon. HhaI and HpaII sites located in the putative CpG island showed a relatively high level of methylation in all the cell populations examined, and did not show any clear correlation with the level of GFAP gene expression or with the methylation status of the −1176 site. Further in vivo developmental studies and in vitro differentiation studies are necessary to better understand the functional differences of the various methylatable CpG sites in the 5′ end of the GFAP gene. © 1994 Wiley‐Liss, Inc.
Effect of persistent mouse hepatitis virus infection on MHC Class I expression in murine astrocytes
Authors: Correale J. , Li S. , Weiner L. P. , Gilmore Wendy
Published: January 2005

Neurotropic strains of mouse hepatitis virus (MHV) have been used extensively for the study of viral pathogenesis in the central nervous system (CNS), serving as models for human neurological diseases such as multiple sclerosis (MS). MHV strains A59 and JHMV both cause acute and chronic encephalomyelitis and demyelination in susceptible strains of mice and rats. In acute disease, CNS damage is most likely the result of lytic infection in neurons and oligodendrocytes, and death can be prevented by the adoptive transfer of Class I‐restricted CD8+ T cells. However, in later stages of the disease induced by some MHV strains, virus tends to be restricted to astrocytes in a nonlytic infection, and the immune response appears to contribute to CNS damage. These data lead us to suggest that the astrocyte may play a central role in the neuropathogenesis of MHV infection. Consistent with this possibility, A59 has been reported to induce the expression of Class I molecules of the major histocompatibility complex (MHC) in glial cells following infection in vivo and in vitro. In this communication, we have examined the influence of persistent infection by both A59 and JHMV on MHC Class I expression in primary murine astrocytes. Persistence was characterized by the presence of intracellular viral antigen and mRNA in the absence of detectable infectious virus particles. Under these conditions, JHMV, but not A59, inhibited constitutive expression of the H‐2 Kb molecule, with the magnitude of inhibition increasing with postinfection time. A59 was not able to induce Class I during persistence, presumably due to the lack of infectious virus particles. Class I expression was restored by the addition of gamma‐interferon (IFN‐γ) to astrocytes persistently infected with either A59 or JHMV. Thus, Class I inhibition is not a permanent consequence of JHMV persistence, and persistence does not interfere with normal signalling pathways for Class I induction. © 1995 Wiley‐Liss, Inc.
Nerve growth factor‐activated protein kinase N modulates the cAMP‐dependent protein kinase
Authors: Volonté C. , Greene L. A.
Published: January 2005

Protein kinase N (PKN) is a serine/threonine protein kinase rapidly activated by nerve growth factor (NGF) and other agents in various cell lines. The possible involvement of PKN in the multiple pathways of the NGF mechanism of action was previously established through the use of purine analogs, some of which are apparently specific inhibitors of this kinase. Since a PKN‐like activity is modulated in several cell lines by cAMP analogs and this activation requires the activity of cAMP‐dependent protein kinase. The aim of the present work is to investigate possible interactions between PKN and C‐PKA. Preincubation of the two kinases in the presence of ATP leads to potentiated phosphorylation of histone HF1, Kemptide (a substrate for C‐PKA, but not for PKN), and several additional substrates. This augmented phosphorylating activity is insensitive to 6‐thioguanine (an inhibitor for PKN, but not for C‐PKA) and is suppressed both by the Walsh inhibitor and by the regulatory subunit of PKA. PKN‐pretreated C‐PKA shows a significant decrease in Km for Kemptide and a substantial increase in Vmax. C‐PKA and PKN are widely expressed enzymes and the possibility of PKN‐dependent modulation of PKA in intact cells would therefore have biological implications for signal transduction mechanisms. © 1995 Wiley‐Liss, Inc.
Morphological and molecular response of the MOCH‐1 oligodendrocyte cell line to serum and interferon‐γ: Possible implications for demyelinating disorders
Authors: Li Y. , Atashi J. , Hayes C. , Reap E. , Hunt Steve , Popko Brian
Published: January 2005

The regional loss of oligodendrocytes is thought to be an important pathological event in a variety of demyelinating diseases of the central nervous system (CNS). Various components of serum, which are normally excluded from the CNS by the blood‐brain barrier, have been implicated as mediators of demyelinating disorders. We have examined the effects of high concentrations of serum (10% fetal bovine serum, FBS), as well as the cytokine interferon‐γ (IFN‐γ), on an oligodendrocyte cell line, MOCH‐1 cells. These cells changed from phase‐bright, small round cells with multiple thin, branched processes in 1% FBS medium to flat, fibroblast‐like cells with large cell bodies when cultured in 10% FBS medium or 1% FBS medium containing IFN‐γ. These morphological changes were accompanied by a large increase in expression of the astrocyte marker, glial fibrillary acidic protein (GFAP), as detected by Northern and Western blot analyses. In addition, Northern blot and fluorescence‐activated cell sorting analyses revealed that IFN‐γ induced a very large increase in major histocompatibility complex (MHC) class I expression in MOCH‐1 cells. MHC class II mRNA induction by IFN‐γ was also seen. In contrast, 10% FBS did not elevate either MHC class I or class II mRNA levels in MOCH‐1 cells. The morphological and molecular effects of 10% FBS and IFN‐γ were reversible. We suggest that the response of MOCH‐1 cells to high concentrations of serum and IFN‐γ may reflect an important in vivo response to oligodendrocytes to perturbations that occur in demyelinating disorders. © 1995 Wiley‐Liss, Inc.
Myelin glycoprotein P0 is expressed at early stages of chicken and rat embryogenesis
Authors: Zhang S.‐M. , Marsh R. , Ratner N. , Brackenbury Robert
Published: January 2005

Although previous studies suggest that P0 is expressed only in myelinating Schwann cells, monoclonal antibody 1E8 reacts with P0, yet also stains early Schwann cell precursors and non‐myelinating Schwann cells (Bhattacharyya et al.: Neuron 7:831–844, 1991). We therefore characterized the 1E8 epitope and analyzed P0 mRNA expression during development. Immunoblot analyses of P0 fusion proteins and of deglycosylated P0 indicated that the 1E8 epitope is polypeptide. Northern blot and polymerase chain reaction (PCR) analyses revealed that P0 is encoded by a single mRNA that is expressed in chicken embryos as early as E4 and in rat embryos as early as E14. These data indicate that the antigen recognized by 1E8 in early chicken embryos is P0 and that, during development of both chickens and rats, P0 mRNA is expressed long before myelination. © 1995 Wiley‐Liss, Inc.
Glial cell line‐derived neurotrophic factor stimulates fiber formation and survival in cultured neurons from peripheral autonomic ganglia
Authors: Ebendal Ted , Tomac A. , Hoffer B. J. , Olson L.
Published: January 2005

Human recombinant glial cell line‐derived neurotrophic factor (GDNF) was tested for its ability to stimulate fiber formation and neuron survival in primary cultures of peripheral ganglia dissected from the chicken embryo. GDNF, first characterized by its actions on central nervous system (CNS) neurons, had a marked stimulatory effect on fiber outgrowth in sympathetic and ciliary ganglia. Weaker responses were evoked in sensory spinal and nodose ganglia and in the ganglion of Remak. In addition, survival of neurons from the sympathetic and ciliary ganglia was stimulated by GDNF at 50 ng/ml. The effects were not mimicked by the distant but related protein transforming growth factor beta 1 (TGFβ1). The profile of neurons stimulated by GDNF is also distinct from the patterns of stimulation shown by nerve growth factor (NGF), stimulation strongly sympathetic but not ciliary ganglia, and ciliary neurotrophic factor (CNTF), stimulating mainly the ciliary ganglion. Moreover, using in situ hybridization histochemistry, GDNF was demonstrated to be present in the pineal gland in the new born rat, a target organ for sympathetic innervation. The present results suggest that GDNF is likely to act upon receptors present in several autonomic and sensory neuronal populations. GDNF may serve to support fiber outgrowth and cell survival in peripheral ganglia, adding yet one more trophic factor to the list of specific proteins controlling development and maintenance of the peripheral nervous system. © 1995 Wiley‐Liss, Inc.
Acetyl‐L‐Carnitine arginyl amide (ST857) increases calcium channel density in rat pheochromocytoma (PC12) cells
Authors: Tewari K. , Simard J. Marc , Peng Y. B. , Werrbach‐Perez K. , Perez‐Polo J. R.
Published: January 2005

We used the patch clamp technique to study the effect of acetyl‐L‐carnitine arginyl amide (ALCAA) and of nerve growth factor (NGF) on availability of L‐type Ca2+ channels in rat pheochromocytoma (PC12) cells maintained in defined medium. Channel availability was measured as number of channels in the patch × the probability of opening (n.P0). In patches from control cells, cells exposed to NGF (10 ng/ml) for six days, and cells exposed to ALCAA (1 mM) for six days, n.P0, measured during 200–240 ms pulses to ‐10 mV (holding potential, −60 mV), was 0.102 ± 0.089 (5 cells), 0.173 ± 0.083 (5 cells), and 0.443 ± 0.261 (7 cells), respectively. The 4.3‐fold increase for the ALCAA‐treated cells was significantly different from control (P < 0.05), whereas that for the NGF‐treated cells was not. For the same conditions, the maximum number of superimposed openings at −10 mV was 1.3 ± 0.5 (6 cells), 1.6 ± 0.5 (8 cells), and 3.3 ± 1.8 (8 cells), with the value for the ALCAA‐treated cells being significantly different from control (P < 0.001). Additional analysis showed that the distribution of channel open times, the time constants, and the voltage dependence of activation were not changed by prolonged exposure to ALCAA. Short‐term exposure to both ALCAA as well as to the parent compound, acetyl‐L‐carnitine (ALCAR), did not cause an increase but rather a decrease in n.P0, and this short‐term effect of both compounds was blocked by neomycin, an inhibitor of phospholipase C. Together, our findings are consistent with the interpretation that short‐term exposure to ALCAA inhibits Ca2+ channel activity, possibly by increasing intracellular Ca2+, and that long‐term exposure causes an increase in Ca2+ channel density, possibly by increasing channel expression, with no change in Ca2+ channel properties. © 1995 Wiley‐Liss, Inc.
Excitatory amino acid release from astrocytes during energy failure by reversal of sodium‐dependent uptake
Authors: Longuemare M. C. , Swanson Raymond A.
Published: January 2005

Non‐synaptic release may be the major route of excitatory amino acid (EAA) efflux during cerebral ischemia. Possible routes of non‐synaptic release include non‐specific anion channels, reversal of Na+‐, CI−‐, or Ca2+‐dependent uptake, and cell lysis. In the present study we employ a novel approach to show reversal of Na+‐dependent uptake as a major route of EAA efflux from astrocyte cultures under conditions of energy failure. Primary rat astrocyte cultures were subjected to combined blockade of glycolytic and oxidative metabolism after incubation with [3H]‐D‐aspartate (D‐ASP). Energy failure produced an efflux of D‐ASP that was maximal by 90 minutes. The efflux over this period was reduced by more than 50% in cells that had been pre‐loaded with PDC (L‐transpyrrolidine‐2,4‐dicarboxylic acid) or TBHA (threo‐β‐hydroxyaspartic acid), compounds that are competitive inhibitors of Na+‐dependent glutamate uptake. The effect of pre‐loading with the inhibitors was concentration dependent. No effect was seen if the inhibitors were added after induction of energy failure, suggesting that the attenuation of D‐ASP efflux resulted from binding of the inhibitors to an intracellular site. These results provide strong evidence that EAA efflux from astrocytes under conditions of energy failure occurs largely through reversal of Na+‐dependent uptake. © 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.
Identification of a second glycine‐like fragment on the strychnine molecule
Authors: Aprison M. H. , Galvez‐Ruano Enrique , Lipkowitz K. B.
Published: January 2005

Strychnine is a complex molecule that inhibits the physiological actions of glycine, an important inhibitory neurotransmitter in the spinal cord, brain stem, and other areas of many vertebrates. Since 1987, we have employed atomistic molecular modeling tools to find an explanation at the molecular level for how this antagonism works. We have located a second glycine‐like fragment in the strychnine molecule that, when compared to glycine in a three pair atom analysis, provides an excellent topological and electronic charge congruence. The topological congruence in the second glycine‐like fragment is much better than with the first fragment reported in 1987 when using a truncated strychnine molecule in the quantum mechanical analysis. A fourth negative atom, a characteristic of antagonists which we reported earlier (Aprison and Lipkowitz: J Neurosci Res 30:442–446, 1991; Aprison and Lipkowitz: J Neurosci Res 31:166–174, 1992) was found in strychnine. This result follows the pattern reported recently for the three weak glycine antagonists N, N‐dimethylmuscimol, N‐methyl‐THIP, and iso‐THAO, a bicyclic 5‐isoxazolol zwitterion. © 1995 Wiley‐Liss, Inc.
Diabetic neuropathy in the rat: 1. Alcar augments the reduced levels and axoplasmic transport of substance P
Authors: Di Giulio A. M. , Lesma E. , Gorio Alfredo
Published: January 2005

This study examined the sciatic nerve axonal transport of substance P‐like immunoreactivity (SPLI) and its basal content in stomach, sciatic nerve and lumbar spinal cord of 8‐ and 12‐week alloxan‐diabetic rats, respectively. One group of diabetic rats received acetyl‐l‐carnitine (ALCAR) throughout the experimental period. Alloxan treatment caused hyperglycemia and reduced body growth. Axonal transport of SPLI was studied by measurement of 24‐hour accumulation at a ligature on the sciatic nerve. There was a marked reduction (from 50% to 100% according to the nerve segment examined) of anterograde and retrograde accumulation of SPLI in the constricted nerve of 8‐week diabetic rats. In the sciatic nerve of ALCAR‐treated diabetic rats, the accumulation of SPLI was comparable to control values. In the sciatic nerve, lumbar spinal cord and stomach of 12‐week diabetic rats, there is a significant reduction of SPLI content. ALCAR treatment prevented SPLI loss in these tissues. Sciatic nerves showed the typical sorbitol increase and myo‐inositol loss that were significantly counteracted by ALCAR. This study suggests that ALCAR treatment prevents diabetes‐induced sensory neuropathy by improving altered metabolic pathways such as polyol activity and myo‐inositol synthesis, and by preventing the reduction of synthesis and axonal transport of substance P. © 1995 Wiley‐Liss, Inc.
Human choline acetyltransferase gene: Localization of alternative first exons
Authors: Chireux M. A. , Le Van Thai A. , Weber Michel J.
Published: January 2005

Two overlapping cosmids containing the 5′ end of human choline acetyltransferase (ChAT) gene have been cloned. Using heterologous probes, we localized two alternative first exons homologous to rodent ChAT exons R and M (Misawa et al.: J Biol Chem 267:20392–20399,1992). The sequence of rodent exon N was not conserved in the human gene. Northern blot analysis of mRNA purified from the human neuroepithelioma cell lines LA‐N2 and MC‐I‐XC revealed that both exons R and M were transcribed in mRNA species of 6.0 and 2.5 kb. Only the 6‐kb species was detected with both R‐ and M‐specific probes in the neuroepithelioma cell line CHP126. Reverse transcription‐polymerase chain reaction (RT‐PCR) analysis suggested that the major mRNA species in MC‐I‐XC and CHP126 cells contained the proximal part of exon M spliced to exon 1, which contains the alternative ACG initiation codon. RT‐PCR also allowed the characterization of a mRNA species containing exon R spliced to exon 1, but no species containing both exon R and the distal part of exon M could be detected. RT‐PCR was also used to evidence an alternative exon (tentatively numbered exon 8) in the coding sequence. © 1995 Wiley‐Liss, Inc.
Complement and cytokine gene expression in cultured microglia derived from postmortem human brains
Authors: Walker D. G. , Kim S. U. , McGeer P. L.
Published: January 2005

Microglia were successfully cultured from human brain tissue from normal and neurologically diseased cases obtained 3.5–10 hours postmortem. Final cell preparations were more than 99% pure as judged by latex bead phagocytosis, expression of microglial phenotypic markers, and absence of astrocytic markers. The expression of complement genes C1qB, C3, and C4 as well as genes for interleukin‐(IL‐)1α, IL‐1β, IL‐6, tumor necrosis factor (TNF)α, IL‐1 receptor antagonist, and transforming growth factor β, but not inducible nitric oxide synthase, by these cells was detected by polymerase chain reaction (PCR) analysis. The pattern of gene expression was evaluated following stimulation of the cells with lipopolysaccharide, phorbol myristate acetate, γ interferon, and β amyloid peptide. There was considerable variation in gene response to these activating agents. However, it was of interest that β‐amyloid peptide (1–40) increased the expression of IL‐1β mRNA in these cells. The number of cases in this study was too small to permit evaluation of microglial response according to the disease state, but the results demonstrate the potential for such studies in the future. © 1995 Wiley‐Liss, Inc.