bluuebunny/crossref_metadata_2025 huggingface dataset:
Long‐term survival of mouse corpus callosum grafts in neonatal rat recipients, and the effect of host sensitization
Authors: Pollack I. F. , Lee L. H.‐C. , Zhou H. F. , Lund R. D.
Published: January 2005

Previous studies have suggested that the incidence of spontaneous rejection among immunogenetically mis matched neural transplants in neonatal recipients varies significantly depending on the cellular composition of the graft material. For example, neuron‐rich grafts of embryonic mouse retina generally survive for extended periods without showing signs of rejection after implantation into neonatal rats, whereas cortical xenografts, which contain abundant glial and endothelial cells as well as neurons, typically undergo rejection 4–6 weeks after implantation. To determine whether the presence of donor glia is responsible for this high incidence of spontaneous rejection, we examined the fate of a non‐neuronal graft material composed predominantly of xenogeneic glial cells (post natal day 3, PD3, CD‐1 mouse corpus callosum) implanted into the mesencephalon of PD1 Sprague‐Dawley rats. The distribution and survival of donor astrocytes were assessed using a monoclonal antibody specific for a mouse astrocyte surface antigen, M2. Thirteen of 16 animals sacrificed within 2 months of implantation had detectable transplants. In these animals, M2‐positive cells frequently migrated well away from body of the graft, clustering in large numbers in several characteristic regions of the host brain. Unlike cortical grafts of similar age, the vast majority (93%) of callosal transplants showed no his‐tological signs of rejection or major histocompatabil‐ity complex antigen expression in and around the transplant‐derived cells. As previously noted in the neonatal retinal transplant paradigm, however, well‐integrated 1‐month‐old corpus callosum grafts could be induced to reject by appropriate sensitization of the host immune system, implying that the host was not immunologically tolerant to the foreign neural graft. With longer survival times in unsensitized hosts, a progressively smaller percentage of animals had detectable donor astrocytes (5 of 10 animals at 3 months postimplantation and 4 of 16 animals at 4 months); in those 9 animals with surviving grafts, only small numbers of M2‐positive cells were seen within the graft bed and surrounding host brain. However, only 2 of the 26 “long‐term” animals showed evidence of graft rejection.

These results indicate that mouse astrocytes show characteristic patterns of migration into the host brain when implanted into neonatal rats; however, these xenogeneic cells have a limited duration of survival. The infrequency with which even subtle signs of spontaneous rejection were detected in animals that had received corpus callosum xenografts suggests that an immune‐mediated process is unlikely to be responsible for the time‐dependent elimination of the donor astrocytes. We conclude that the presence of donor astrocytes does not itself predispose a graft to rejection. Accordingly, these cells are unlikely to be responsible for the high incidence of spontaneous rejection seen with cortical grafts.
Calmodulin antagonists chlorpromazine and W‐7 inhibit exogenous cholesterol esterification and sphingomyelinase activity in human skin fibroblast cultures. Similarities between drug‐induced and niemann‐pick type C lipidoses
Authors: Masson M. , Spezzatti B. , Chapman J. , Battisti C. , Baumann N.
Published: January 2005

In this report we showed that calmodulin antagonists chlorpromazine (CPZ) and W‐7 (N‐[6‐ aminohexyl]‐5‐chloro‐1‐naphtalenesulfonamide), when added to fibroblast cell cultures, gave rise to a time‐ and dose dependent decrease of sphingomyelinase activity. CPZ and W‐7 also significantly inhibited LDL‐ and non‐LDL‐ dependent cholesterol esterification. Addition of these drugs to cell culture medium mimicked what is observed in the genetic disease Niemann‐Pick type C.H‐7 (1‐[5‐isoquinonylsulfonyl]‐2‐methylpiperazine), an inhibitor of protein kinase C and cyclic nucleotide‐dependent kinases, had no effect on sphingomyelinase and cholesterol ester formation. Thus the possibility of a modulation of cell sphingomyelin and cholesterol esters by a calmodulin‐dependent second messenger system must be considered.
Expression of the neu oncogene under the transcriptional control of the myelin basic protein gene in transgenic mice: Generation of transformed glial cells
Authors: Hayes C. , Kelly D. , Murayama S. , Komiyama A. , Suzuki K. , Popko B.
Published: January 2005

We have taken a transgenic approach in an effort to specifically transform oligodendrocytes, the myelinating glial cells of the central nervous system (CNS). Transgenic mice were generated with a DNA construct that contained the activated neu oncogene under the transcriptional control of the myelin basic protein (MBP) gene. The MBP/c‐neu transgenic animals have experienced a low incidence of brain tumors that express molecular markers specific to oligodendrocytes, providing a mouse model to study the formation and progression of oligodendrocyte tumors. A tumor from a transgenic animal has been dispersed in culture, and transformed cells that express properties of oligodendrocytes and astrocytes have been maintained. The degree to which these cells express phenotypic characteristics of oligodendrocytes or astrocytes is influenced by culture conditions. These transformed cells should serve as a valuable resource with which to study various molecular and biochemical aspects of the myelination process, as well as the lineage interrelationship of CNS glial cells.
Axons modulate the expression of proteolipid protein in the CNS
Authors: Scherer Steven S. , Vogelbacker H. H. , Kamholz J.
Published: January 2005

We examined the expression of mRNA encoding proteolipid protein (PLP), the major myelin protein in the CNS, in developing rat cerebrum, and in normal and degenerating optic nerves. PLP transcripts were initiated at two clusters of start sites that were separated by 30 base pairs. During the peak of PLP mRNA expression in developing cerebrum, a higher proportion of PLP transcripts were initiated from the destal start site, furthest from the open reading frame, than in mature cerebrum. We enucleated one eye of immature rats to cause Wallerian degeneration in the optic nerve. In these degenerating optic nerves, the steady state levels of PLP mRNA fell markedly, and the proportion of distally initiated PLP transcripts declined to the same proportion found in normal adult nerves. Changes in myelin gene expression were not limited to PLP mRNA, as the steady‐state levels of myelin basic protein (MBP) mRNA paralleled those of PLP mRNA in the developing cerebrum and in degenerating optic nerves. Thus, oligodendrocytes require axons to maintain their normal levels of PLP and MBP transcripts and the high proportion of distally initiated PLP transcripts that characterize early myelination. © 1992 Wiley‐Liss, Inc.
Cyclic AMP, but not basic FGF, increases the in vitro survival of mesencephalic dopaminergic neurons and protects them from MPP+‐induced degeneration
Authors: Hartikka J. , Staufenbiel M. , Lübbert H.
Published: January 2005

We studied how stimulation of protein kinase C and cAMP‐dependent protein kinases affect the development of mesencephalic dopaminergic neurons in primary cell cultures derived from fetal rats at embryonic day E14. The effects of compounds which activate these second messenger systems were compared to those of basic fibroblast growth factor (bFGF) and insulin‐like growth factor I (IGF‐I). In mesencephalic cultures, there was a continuous loss of dopaminergic neurons. Despite this decline in cell number, neurotransmitter uptake per neuron increased with time, indicating that the surviving dopaminergic neurons continued their biochemical differentiation while others degenerated. IGF‐I and bFGF did not affect the number of dopaminergic neurons. However, dopamine uptake per neuron was significantly higher in bFGF and IGF‐I treated cultures, suggesting that these factors stimulated differentiation. Protein kinase C and cAMP‐dependent protein kinases were not involved in mediating the effects of bFGF and IGF‐I. Treatment of cultures with phorbol esters did not affect dopamine uptake, whereas elevated levels of intracellular cAMP resulted in an increase in dopamine uptake which was additive to that elicited by bFGF or IGF‐I. Further analysis revealed that exposure of mesencephalic cultures to dibutyryl cAMP (dbcAMP) during the first 3 days after plating increased the survival of dopaminergic neurons, whereas prolonged treatment attenuated the development of the dopamine uptake system. Moreover, cyclic AMP, but not bFGF, was able to prevent the degeneration of dopaminergic neurons induced by 1‐methyl‐4‐phenyl‐pyridinium ion (MPP+), the active metabolite of 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP). The results suggest that increased intracellular levels of cAMP protect dopaminergic neurons in situations of stress like the process of dissociation and plating or the exposure to neurotoxic compounds. Our results reveal novel possibilities for the treatment of Parkinson's disease. © 1992 Wiley‐Liss, Inc.
Immunohistochemical and biochemical analysis of N‐cadherin expression during CNS development
Authors: Lagunowich L. A. , Schneider J. C. , Chasen S. , Grunwald Gerald B.
Published: January 2005

The expression of the calcium‐dependent adhesion molecule N‐cadherin during chick embryo central nervous system (CNS) development was examined by immunohistochemistry and electrophoresis and immunoblotting. During histogenesis, N‐cadherin is expressed at high levels in a uniform fashion in many regions of the CNS. However, during later stages of development, expression becomes restricted to the ependymal cells lining the ventricular system and in the choroid plexus. This down‐regulation was confirmed by both immunohistochemical and biochemical techniques. The program of expression lags behind in the cerebellum in concert with the delayed development of this region of the brain. A high level of N‐cadherin was found to be expressed in the brainstem and spinal cord floorplate, while a low level was detected at the optic nerve head. The results indicate that while, in general, the program of N‐cadherin expression is similar in the retina and the brain, certain structures unique to the eye and brain express locally high or low levels of this adhesion protein. © 1992 Wiley‐Liss, Inc.
Effect of a discrete dorsal forebrain lesion in the rat on the expression of neuronal and glial‐specific genes: Induction of calmodulin, NF‐L, SC1, and GFAP mRNA
Authors: Landry C. F. , Ivy G. O. , Brown Ian R.
Published: January 2005

The influence of a localized tissue injury on the expression of genes encoding specific neuronal and glial proteins was examined using in situ hybridization. A pronounced induction of neuron‐specific calmodulin (CaM) mRNA was evident within cells proximal to the wound site by 2 hours following a cortical lesion in rats. By 12 hours postlesion, intense signal corresponding to CaM mRNA was found to extend 1 mm from the wound site. Changes in the expression of mRNA encoding two additional neuronal proteins, the 68 kilodalton neurofilament protein and the extracellular matrix protein, SC1, were also evident at 12 hours following the cortical injury. Of the two glial proteins examined, a dramatic elevation in levels of mRNA for glial fibrillary acidic protein (GEAP) was observed at the wound site by 12 hours postlesion. This intense labeling corresponding to GFAP mRNA was evident in the ipsilateral glial limitans and hippocampus as well as in the contralateral glial limitans. In contrast, the pattern of labeling for the β‐subunit of the S100 protein did not differ from that of control animals at either of the postlesion intervals examined. This study identifies four genes, CaM, GFAP, SC1, and NF‐L, which are induced following a localized cortical injury and which encode mRNA species enriched in specific cell‐types in the central nervous system. © 1992 Wiley‐Liss, Inc.
AP‐1 complex and c‐fos transcription are involved in TPA provoked and trans‐synaptic inductions of the tyrosine hydroxylase gene: Insights into long‐term regulatory mechanisms
Authors: Icard‐Liepkalns C. , Biguet N. Faucon , Vyas S. , Robert J. J. , Sassone‐Corsi P. , Mallet J.
Published: January 2005

We have previously shown that the phorbol ester, TPA, which activates protein kinase C, causes, in PC12 cells, a transcriptional activation of tyrosine hydroxylase (TH), the key enzyme in catecholamine synthesis. The study has now been extended to examine the processes that underlie this transcriptional stimulation and, in addition, to seek whether similar mechanisms are involved in long‐term trans‐synaptic induction of the TH gene in adrenal medullae of rats that have been given a single injection of reserpine.

In both systems, it was found that the induction of c‐fos gene transcription was associated with that of the TH gene but with different kinetics. The promoter of the TH gene contains (at position −207/–200) a sequence (TGATTCA) which differs from the consensus TRE or AP‐1 site (TGACTCA) by one nucleotide. Experiments were carried out to investigate whether the AP‐1 protein complex which is known to contain Fos and Jun binds to the putative TRE region of the TH promoter. In the gel shift assays, the nuclear protein extracts derived from TPA‐treated PC12 cells and from AM of reserpine injected rats displayed a higher magnitude of binding to a 25‐mer TRE‐TH oligonucleotide as compared to controls. The results showed that the behaviour of TRE‐TH was atypical in that two retarded complexes (A and B) were observed, which were displaced by specific competitors. Trans‐activation experiments with plasmids TRE‐TH/TK/CAT and −754/–19 TH/pUC18‐CAT in PC12 cells showed an increase in CAT activity in response to TPA that correlates with the previously observed increase in TH transcriptional activity by TPA. Co‐transfection with plasmids BK28 containing c‐fos and/or with pSV‐c‐jun resulted in a significant increase in CAT activity showing a functional interaction of these proteins with TRE‐TH. Our findings indicate that the TRE‐TH site plays a critical role in trans‐synaptic regulation of the TH gene. © 1992 Wiley‐Liss, Inc.
Basic fibroblast growth factor in the hypoglossal system: Specific retrograde transport, trophic, and lesion‐related responses
Authors: Grothe Claudia , Unsicker K.
Published: January 2005

To further clarify the function of basic fibroblast growth factor (bFGF) in the nervous system, we have examined its distribution, lesion‐dependent regulation, retrograde transport, and trophic roles on rat hypoglossal neurons. In adult rats, bFGF‐like immunoreactivity is localized in hypoglossal motoneurons, drastically reduced 2 days after axotomy, and re‐expressed by 11 days. Neuron numbers and morphology assessed by Nissl staining are not affected by the lesion. 125J bFGF is specifically retrogradely transported by hypoglossal motoneurons from their peripheral nerve terminals. Moreover, bFGF stimulates the in vitro survival of hypoglossal neurons (ED50 2 ng/ml). In vivo administration of bFGF prevents lesion‐induced motoneuron death to 14% in 7 day old rats and to 60% in 18 day old rats, but not the axotomy‐induced decrease of choline acetyltransferase activity in the hypoglossal nucleus of adult rats. These results are consistent with a neurotrophic role of bFGF in the hypoglossal system. © 1992 Wiley‐Liss, Inc.
Two‐site enzyme immunoassay for βNGF applied to human patient sera
Authors: Lorigados L. , Söderström S. , Ebendal T.
Published: January 2005

Nerve growth factor (NGF) supports sympathetic and sensory neurons in the peripheral nervous system and serves functions in the development and maintenance of cholinergic neurons in the basal forebrain. NGF distribution can be studied with the use of a sensitive two‐site enzyme immunoassay (EIA). The monoclonal antibody 27/21 to mouse NGF was recently shown to effectively block the activity of both recombinant human NGF and native mouse NGF, and a two‐site EIA using monoclonal antibody 27/21 was optimized. We have now applied this assay to examine NGF levels in normal human serum and serum from Parkinson, Alzheimer, and Huntington patients. To further test the specificity of conjugate binding, dilutions of the human sera were preincubated with an excess of monoclonal NGF antibody 27/21 in solution. With this strategy it was possible to completely block the signal obtained using the two‐site EIA. Furthermore, we show that recombinant BDNF and NT‐3 do not cross‐react with monoclonal antibody 27/21 under our conditions. We found low levels of specific NGF immunoreactivity in normal human sera (0.4 ± 0.1 ng/ml). Significantly lower levels of NGF were found in sera from patients with Parkinson's and Huntington's disease whereas sera from Alzheimer patients showed only slight reductions in the NGF level. Two patients who had received intracerebral NGF infusions (one with Parkinson's and other with Alzheimer's disease) showed significantly elevated serum levels of NGF during the period of infusion. Due to an inhibitory activity in human serum, it was impossible to demonstrate the low levels of NGF activity in the human serum samples using explanted embryonic sympathetic ganglia, even after concentration by pressure dialysis. Thus, the serum levels are below the limit to evoke a response in NGF‐sensitive neurons and thus to expect any physiological effect. Nevertheless, the levels measured may be used as indicators in clinical conditions such as Parkinson's and Huntington's disease. © 1992 Wiley‐Liss, Inc.
Receptor neuron losses result in decreased cytochrome P‐450 immunoreactivity in associated non‐neuronal cells of mouse olfactory mucosa
Authors: Walters E. , Buchheit K. , Maruniak J. A.
Published: January 2005

Immunohistochemical and immunoblot analyses were used to investigate nasal cytochrome P‐450 distribution in mice with either unilateral naris closure for 3, 4, or 5 months, or olfactory bulbectomy. P‐450 immunoreactivity was observed only in the supporting cells and Bowman's glands of the olfactory mucosa. Immunoreactivity was clearly reduced in rostral regions of the open‐side olfactory mucosa where losses of receptor neurons resulted from 3 to 5 months of closure. Closed‐side immunoreactivity was similar to controls. In 4 month closure animals that had regrown their receptor neurons, open‐side immunoreactivity was comparable to controls. Olfactory bulbectomy also depressed P‐450 immunoreactivity. These data suggest that presence or absence of receptor neurons markedly affects P‐450 expression in nonneuronal cells of the olfactory mucosa. © 1992 Wiley‐Liss, Inc.
Taurine allosterically modulates flunitrazepam binding to synaptic membranes
Authors: Quinn M. R. , Miller C. L.
Published: January 2005

Taurine is hypothesized to exert its inhibitory neuromodulatory effects, in part, by interaction with the GABAA receptor. Although taurine displaces GABA agonist binding to synaptic membranes, its allosteric effects on the benzodiazepine recognition site of the GABAA receptor complex is unsettled. We determined the effects of taurine on [3H]flunitrazepam (Flu) binding to well‐washed, frozen‐thawed synaptic membranes prepared from rat cortex. Comparative binding studies were conducted at 37°C and on ice (0–4°C). At 37°C taurine increased Flu binding in a concentration dependent way by interaction with a bicuclulline sensitive site, similar to GABA. Taurine increased Flu binding by causing a decrease in KD. The maximal effectiveness of taurine on Flu binding could not be increased further by addition of GABA. In contrast, the maximal stimulation of Flu binding by GABA was decreased by addition of taurine to the level attained by taurine alone. These mixed agonist/antagonist effects of taurine are pharmacologically specific and qualify taurine as a partial GABA agonist in this type of allosteric interaction. However, taurine causes opposite effects on Flu binding when measured at 0–4°C: taurine interacts with a bicuculline insensitive site to inhibit Flu binding by increasing the KD. Taurine inhibition of Flu binding is not overcome by increasing concentrations of GABA. Although the mechanism of taurine inhibition of Flu binding at 0–4°C is unclear, it may be an indirect effect of taurine interaction with membrane phospholipids. More importantly, the results of these studies suggest that taurine, at physiologically relevant concentrations (EC50 = 100 μM) and temperature (37°C), may function as a partial GABA agonist in stimulating Flu binding to synaptic membranes. © 1992 Wiley‐Liss, Inc.
Gene structure and expression of the mouse 5‐HT2 receptor
Authors: Yang W. , Chen K. , Lan N. C. , Gallaher T. K. , Shih J. C.
Published: January 2005

Serotonin (5‐Hydroxytryptamine, 5‐HT) is an important neurotransmitter which mediates numerous physiological functions. Using the SacI‐EcoRI restriction fragment of the rat brain 5‐HT2 receptor cDNA as a probe, we have screened a mouse brain cDNA library, created by random priming and constructed in SWAJ vectors, and have isolated a cDNA encoding a 1.4 kb open reading frame which codes for a functional mouse 5‐HT2 receptor identified from pharmacological binding profiles and coupling of phosphoinositide formation in a stably transfected fibroblast cell line. The deduced amino acid sequence is 97.4% identical to the rat 5‐HT2 receptor. Using the same 5‐HT2 receptor cDNA probe, ten positive genomic clones were isolated from two mouse genomic libraries constructed in the pWE15 cosmid vector and the EMBL‐3 phage vector. Extensive mapping and sequencing of these genomic clones indicate the mouse 5‐HT2 receptor coding region spans over 20 kb and is composed of three exons split by two introns. Northern blot analysis shows one band of 5–6 kb in the mouse brain, but not in the heart, lung, liver, or kidney total RNA. Southern analysis of mouse liver genomic DNA shows a simple pattern of digestion by several restriction enzymes, which suggests that one copy of the 5‐HT2 receptor gene may exist in the mouse genome. © Wiley‐Liss, Inc.
Effect of potassium and N‐methyl‐D‐aspartate on the aspartate aminotransferase activity in cultured cerebellar granule cells
Authors: Morán J. , Rivera‐Gaxiola M.
Published: January 2005

The effect of potassium depolarization and N‐methyl‐D‐aspartate (NMDA) on the activity of aspartate aminotransferase (AAT; EC 2.6.1.1), an enzyme suggested to be involved in neurotransmitter glutamate synthesis, was studied in cultured cerebellar granule neurons. Both KCl and NMDA increased AAT activity in a dose‐dependent manner. When cells were treated 48–72 hr with 40 mM KCl or 150 μM NMDA the AAT was enhanced about 65–75%. The EC50 for NMDA and KCl were 25 μM and 17 mM, respectively. The effect of NMDA and KCl was specific for AAT without affecting the activity of other enzymes like lactate dehydrogenase or protein content and it was observed only in granule cells but not in astrocytes or cortical neurons. The effect of KCl was not mediated by an activation of excitatory amino acid receptors and was Ca++‐dependent. The effect of NMDA was completely blocked by Mg++ and NMDA antagonists. The increase of AAT induced by AAT and KCl was blocked by cycloheximide and actinomycin D, suggesting an involvement of de novo synthesis of proteins and RNA. Kainic acid and quinolinic acid were also effective in increasing the AAT activity. The action of kainate was less effective than that of NMDA and it was observed only at relatively low concentrations (10 μM). Quinolinic acid raised the activity of AAT about 45% at a concentration of 500 μM. Other non‐NMDA agonists did not modify the AAT activity. From these findings we can conclude that NMDA and KCl exert a trophic action on cerebellar granular neurons. These conditions could be a simulation of the presynaptic activity required during development to induce survival and a biochemical differentiation, which we evaluated using AAT as a marker enzyme. © Wiley‐Liss, Inc.
Uptake and metabolism of malate in neurons and astrocytes in primary cultures
Authors: Hertz L. , Yu A. C. H. , Schousboe A.
Published: January 2005

Uptake and oxidative metabolism of [14C]malate as well as its incorporation into aspartate, glutamate, glutamine, and GABA were studied in cultured cerebral cortical neurons (GABAergic), cerebellar granule neurons (glutamatergic), and cerebral cortical astrocytes. All cell types exhibited high affinity uptake of malate (Km 10–85 μM) with slightly higher Vmax values in neurons (0.1–0.2 nmol × min−1 × mg−1) than in astrocytes (0.06 nmol × min−1 × mg−1). Malate was oxidatively metabolized in all three cell types with nominal rates of 14CO2 production of 2–15 pmol × min−1 × mg−1. The oxidation of malate was only slightly inhibited by 5 mM aminooxyacetic acid (AOAA). In granule cell preparations [14C]malate was incorporated into aspartate and glutamate and, to a much less extent, into glutamine. This incorporation was blocked by 5 mM AOAA. Astrocytes exhibited slightly higher incorporation rates into aspartate and glutamate, but in these cells glutamine was labelled to a considerable extent. AOAA (5 mM) inhibited the incorporation by 60–70%. In cultures of cerebral cortical neurons, very low levels of radioactivity derived from [14C]malate were found in aspartate and glutamate, and GABA was not labelled at all. Glutamine had the same specific activity as glutamate, indicating that the low rates of incorporation of radioactivity into amino acids in this preparation is likely to exclusively represent metabolism of malate in the small population of astrocytes (5% of total cell number), contaminating the neuronal cultures. The findings suggest that exogenous malate to a quantitatively limited extent may serve as a precursor for transmitter glutamate in glutamaterigc neurons. Astrocytes are able to metabolize malate to glutamate and related amino acids, but oxidize little malate to CO2. © Wiley‐Liss, Inc.
Interaction of ganglioside GM1 with the B subunit of cholera toxin modulates intracellular free calcium in sensory neurons
Authors: Milani D. , Minozzi M.‐C. , Petrelli L. , Guidolin D. , Skaper S. D. , Spoerri P. E.
Published: January 2005

The B subunit of cholera toxin, which binds specifically to GM1 ganglioside on cell surfaces, has previously been shown to modulate intracellular calcium levels and growth in several cell types. To explore a role for such changes in calcium in the growth regulatory function of cell‐associated GM1 in neurons, dissociated neurons from chicken embryonic day 8 dorsal root ganglia were exposed to the B subunit. To enhance sensitivity to B subunit, some neurons were also enriched with added GM1 (100 μM) and then exposed to B subunit. Incubation of naive cultures with 1 μg/ml of the B subunit was sufficient to produce modest increases in intracellular free calcium above basal levels in a minor percentage of cells for at least 5 min, as measured by fura‐2 fluorescence imaging. Pretreatment of the cells with GM1 for 48 hr increased even further the elevations in intracellular free calcium and the percentage of responding neurons observed after B subunit exposure. These in creases in intracellular calcium required the presence of external Ca2+, but were not inhibited by calcium channel blockers. Such changes in calcium were accompanied by fine alterations in morphology affecting mostly the branching of neurites and were more pronounced in the presence of GM1. However, the morphological changes did not result in altered neurofilament protein expression. Immunogold electron microscopy using anti‐choleragenoid depicted extensive aggregations of immunoreactive gold particles on neuronal surfaces, which were more extensive in cells treated with GM1. The results demonstrate that cell incorporated GM1 may modulate calcium fluxes, perhaps accounting for the growth regulatory functions of GM1 in both neuronal and other cell types. © 1992 Wiley‐Liss, Inc.
Treatment of relapsing experimental autoimmune encephalomyelitis with T cell receptor peptides
Authors: Whitham R. H. , Kotzin B. L. , Buenafe A. C. , Weinberg A. D. , Jones R. E. , Hashim G. A. , Hoy C. M. , Vandenbark A. A. , Offner H.
Published: January 2005

Restricted T cell receptor (TCR) VB gene usage by T cells for recognition of antigens involved in the production of experimental autoimmune encephalomyelitis (EAE) offers the possibility of selective immunotherapy. We determined the preferential VB gene usage of lymph node‐derived clones from SJL/J mice to recognize the encephalitogenic epitope PLP 139‐151 and from PL/J mice to recognize the newly described encephalitogenic epitope PLP 43‐64. In addition, the VB gene usage for recognition of PLP 139‐151 by T cell lines derived from SJL/J spinal cords was analyzed. Lymph node‐derived SJL/J lines and clones specific for PLP 139‐151 expressed VB2, VB4, and VB17a preferentially, and PL/J lines and clones specific for PLP 43‐64 expressed VB2 and VB8.2 preferentially. A VB4+ SJL/J clone and a VB8.2+ PL/J clone were encephalitogenic. Encephalitogenic SJL/J lines derived from spinal cord expressed VB2, VB10, VB16, and VB17a preferentially, with a predominance of VB2. Candidate TCR peptides were synthesized and tested from the VB gene families VB4, VB8.2, and VB17a, based on our data and previous data on BP‐induced EAE in mice. Treatment of relapsing EAE (R‐EAE) in SJL/J mice with VB4 and VB17a peptides reduced clinical and histological disease severity, and treatment of R‐EAE in (PLxSJL)F1 mice with VB4 and VB8.2 peptides also reduced clinical and histological disease. The use of TCR peptide therapy may have applications for the treatment of human autoimmune diseases such as multiple sclerosis. © 1993 Wiley‐Liss, Inc.
Sp1 binds to the adhesion molecule on glia regulatory element that functions as a positive transcription regulatory element in astrocytes
Authors: Kawakami K. , Watanabe Y. , Araki M. , Nagano K.
Published: January 2005

We analyzed cis‐acting elements regulating the expression of the gene encoding adhesion molecule on glia (AMOG) in primary cultured astrocytes from newborn rat cerebrum and cerebellum. The relative promoter activities among the series of 5‐ sequential deletion mutants are similar to those observed in B103 (rat neuroblastoma cell line) cells. The previously identified AMRE (AMOG regulatory element) of the GAGGCGGGG sequence functions as a positive regulatory element, not only in B103 cells, but also in astrocytes. Binding factors to the element were identified as Sp1 based on the following observations using nuclear extracts from the astrocytes and B103 cells: (1) The interaction of the factors with AMRE analyzed by DNase I footprinting and methylation interference analyses was similar to that of Sp1; (2) The binding of the factors to AMRE competed with an oligonucleotide containing the authentic Sp1 consensus sequence; (3) Sp1‐specific antibody interfered with the formation of the AMRE gel retardation complexes. The functional implications of the factors in AMOG gene regulation are discussed. © 1993 Wiley‐Liss, Inc.
Elevated expression of B‐50 (GAP‐43)‐mRNA in a subpopulation of olfactory bulb mitral cells following axotomy
Authors: Verhaagen J. , Zhang Y. , Hamers F. P. T. , Gispen W. H.
Published: January 2005

Neurons in the central nervous system regenerate poorly or not at all. In contrast neurons of the peripheral nervous system have the ability to regrow their nerve fibers over considerable distances. Previously it has been suggested that the absence of the reinduction of the expression of growth associated proteins such as B‐50 (GAP43) may be an important factor in the differential response of CNS and PNS neurons to injury. We studied B‐50(GAP43) mRNA expression following lesioning of a class of CNS neurons, the olfactory bulb mitral cells. Expression of B‐50 mRNA in approximately 40% of the mitral cells was upregulated in response to transection of their axons in the lateral olfactory tract (LOT). Enhanced expression persisted for 10 days postlesion but had virtually declined to control levels by 4 weeks after the lesion. A large proportion of the mitral cells gradually degenerated subsequent to LOT transection. Thus a subpopulation of mitral cells maintains their ability to upregulate B‐50, a protein characteristic of growing axons, but enhanced B‐50 expression is not accompanied by regeneration of the severed LOT. © 1993 Wiley‐Liss, Inc.
Development of NMDA receptor‐channel complex and L‐type calcium channels in mouse hippocampus
Authors: Glazewski S. , Skangiel‐Kramska J. , Kossut M.
Published: January 2005

In vitro binding autoradiography was used to examine the pattern and intensity of binding of [3H]glutamate to NMDA receptors, [3H]MK 801 to NMDA receptor associated channels in and [3H]PN‐200 110 to L‐type calcium channels in the hippocampus of mice aged 3–70 days. The distribution of NMDA receptors and NMDA receptor associated channels was similar but not identical at the tested ages. Beginning with postnatal day 8, high binding levels were confined mostly to the hippocampal strata: the oriens and radiatum (CA1 and CA3 with [3H]MK 801 labeling but only CA1 with NMDA displaced [3H]glutamate labeling), the moleculare (higher labeling with [3H]MK 801 than with NMDA displaced [3H]glutamate binding), and the lucidum. The binding values for NMDA receptor‐channel complex rose in the examined period (especially within the second and third week), reaching a plateau at the end of the third postnatal week. Sharp growth of binding within the second and third week of life was about 50% greater with [3H]MK 801 than with NMDA displaced [3H]glutamate labeling. L‐type calcium channels were found to be most abundant in the strata: the oriens of the CA3 field, the moleculare, and the lucidum. The time course of binding value changes for the calcium channel was similar to the time course found for the NMDA receptor‐channel complex. © 1993 Wiley‐Liss, Inc.
Nerve growth factor changes G protein levels and localization in PC12 cells
Authors: Zubiaur M. , Neer E. J.
Published: January 2005

Growth cones at the growing tips of developing neurites contain the machinery to transmit information from receptors to a variety of intracellular enzymes and ion channels. In order to understand how signals are transmitted across the membrane, we asked whether the multiplicity of signalling pathways in the growth cone is reflected by the diversity of G proteins found in this organelle. Our immunohistochemical analysis indicated that growth cones of differentiated PC12 cells contain at least 4 α G protein subunits, 3 that are pertussis toxin substrates (α0, αi−1, αi−2) and 1 that is not (αq). In addition to localization in the neurites and growth cones, α0, αi−1, αi−2, and αq were detected in intracellular perinuclear structures. We also analyzed the temporal change in G proteins in PC12 cells differentiated by treatment with nerve growth factor (NGF). Time course experiments have shown that α0 and β proteins coordinately increase after 2 days of treatment with NGF, reach a maximum at 4 days, and remain elevated. In contrast to α0, αi−2 reached a peak at 4 days, then declined to almost the basal level by day 7 of treatment with NGF. These data indicated that the levels of α0, αi−2, and β are differentially regulated during NGF‐induced neuronal differentiation in PC12 cells. The α0 protein was highly concentrated at the tips of the growth cones before the cellular level of α0 had increased appreciably, suggesting that the α subunits are translocated during the first stage of neurite development. In addition, not every neural process has the same high level of α0, suggesting that G proteins may help define the specialized functions of particular neurites within a single cell. © 1993 Wiley‐Liss, Inc.
Maternal dietary fish oil enriches docosahexaenoate levels in brain subcellular fractions of offspring
Authors: Yeh Y.‐Y. , Gehman M. F. , Yeh S.‐M.
Published: January 2005

Prompted by the speculated essentiality of docosahexaenoic acid (DHA) for neural development, this study was undertaken to investigate the incorporation of (n‐3) fatty acids in the maternal diet into various phospholipids of infant rat brain subcellular fractions: microsomes (Ms), synaptosomes (Sy), myelin (My), and mitochondria (Mt). Two groups of infant rats were nourished by dams fed diets containing 20% of either corn oil (CO) or menhaden oil (MO) from 2 until 12 days of age. DHA but not eicosapen taenoic acid (EPA) was distributed to all subcellular fractions of infant rats in the CO group. The levels of DHA were higher in Ms and Mt than Sy and My, and higher in phosphatidylethanolamine (PE) and phosphatidylserine (PS) than phosphadylcholine (PC) and phosphatidylinositol (PI). The MO feeding enriched DHA in PE of all subcellular fractions, PS of all subcellular fractions, except My, PC of Sy, My and Mt, and PI of My. EPA was enriched in phospholipids in all subcellular fractions, except mitochondrial PS of the MO group. In the MO group, the ratios of EPA/ DHA, ranging from 0.01 to 0.85, in all subcellular phospholipids were markedly lower than that found in the mother‐s milk (i.e., 1.5), suggesting an ability to elongate and desaturate EPA to DHA and/or disproportional uptake of the fatty acids by the brain. In PE of all subcellular fractions, the increased levels of DHA and EPA, with a concomitant reduction of arachidonic and/or linoleic acid, yielded higher ratios of total (n−3)/(n−6) fatty acids in the MO than the CO group. The inclusion of preformed DHA and EPA in the maternal diet provides an effective means to enrich these fatty acids in developing brains. © 1993 Wiley‐Liss, Inc.
Testosterone regulation of the regenerative properties of injured rat sciatic motor neurons
Authors: Kujawa K. A. , Jacob J. M. , Jones K. J.
Published: January 2005

We have previously demonstrated that systemic administration of testosterone differentially regulates the regenerative properties of injured hamster facial motor neurons, which are androgen receptor‐containing cranial motor neurons. In this investigation the hypothesis that testosterone alters the regenera‐ tive properties of rat sciatic motor neurons, which are androgen receptor‐containing spinal motor neurons, was tested using fast axonal transport of radioactively labeled proteins to assess sciatic nerve regeneration. Adult castrated male rats were subjected to crush axotomy of the sciatic nerve at the level of the gemelli tendons (mid‐thigh). One‐half of the axotomized animals received subcutaneous implants of testosterone propionate (TP), with the remainder of the animals sham implanted with blank capsules. The outgrowth distances of the leading axons were measured at 5, 6, 7, and 11 days postoperative. Linear regression analysis was accomplished, with the slope of the line representing the regeneration rate and the x‐intercept the initial delay of sprout formation. Systemic administration of testosterone resulted in a 13% increase in the rate of regeneration, relative to the control, –TP group. Outgrowth distances were significantly increased in the 4‐ TP group only in the later stages of regeneration. However, TP did not shorten the delay in sprout formation in regenerating sciatic motor neurons, but instead produced a small prolongation in the delay time. This pattern of hormonal regulation of the regenerative properties of spinal motoneu‐ rons is similar to that previously found in cranial motoneurons. The prolongation of the initial delay may have been a factor in the lack of significant outgrowth distances during the early stages of regeneration. The magnitude of the effects of TP on male rat sciatic motor neurons are less pronounced than in male hamster facial motor neurons, but quite similar to those effects observed in female hamster facial motor neurons. Why this is the case is not known, but may be related to a irumber of factors discussed in the text. © 1993 Wiley‐Liss, Inc.
Cerebellar and striatal dopamine receptors: Effects of reeler and weaver murine mutations
Authors: Panagopoulos N. T. , Matsokis N. A. , Valcana T.
Published: January 2005

The presence and the binding characteristics of D1 and D2 receptors were investigated in normal‐reeler and normal‐weaver mutant mice utilizing [3H]spiperone (D2 antagonist), [3H]SKF 38393 (D1 agonist), and [3H]DA as ligands. Analysis of the binding data showed that in the cerebellum there are two binding components for all [3H]ligands. Comparison of the binding constants from cerebellum and striatum showed that in cerebellum the high affinity‐low capacity component has similar affinity with that of striatum. The reeler and weaver mutations affected the binding of all ligands: In reeler, total cerebellar specific binding sites for [3H]spiperone and [3H]SKF 38393 decrease significantly (≈50% and ≈70%, respectively), while those for [3H]DA show a small (≈10–15%) but not significant decrease. In weaver, total cerebellar specific binding sites for [3H]spiperone, [3H]SKF 38393, and [3H]DA also decrease significantly (≈60%, ≈70%, and ≈50%, respectively). In reeler striatum [3H]SKF 38393 binding (Bmax) is significantly decreased (≈24%), while [3H]spiperone and [3H]DA binding (Bmax) is not affected. In weaver striatum, [3H]SKF 38393 binding is significantly increases significantly (≈40%), while [3H]DA binding (Bmax) decreases significantly (≈70%). On the basis of the cytoarchitectural aberrations that characterize the cerebellum of these mutants and some well‐established information regarding the dopaminergic system of the cerebellum, the above results indicate that in this region (a) D1 receptors are mainly localized on granule cells and (b) D2 receptors are localized postsynaptically on granule cells and presynaptically on the DA fibers innervating the cerebellum. © 1993 Wiley‐Liss, Inc.
Molecular cloning of the myelin basic proteins in the shark, Squalus acanthias, and the ray, Raja erinacia
Authors: Spivack W. D. , Zhong N. , Salerno S. , Saavedra R. A. , Gould R. M.
Published: January 2005

Myelin basic proteins (MBPs) are a family of alternatively spliced isoforms present in myelin sheaths of most vertebrates. A reverse transcriptase‐polymerase chain reaction (RT‐PCR) approach was used to clone MBP isoforms in species representing two superorders of elasmobranchs: Squalus acanthias, representing Squalomorph sharks, and Raja erinacia, representing Batoidea rays. Two products were generated from each species. The larger product encoded a 155 amino acid protein, the same size as MBPs from two Galeomorph sharks, Heterodontus francisci and Carcharhinus obscurus, which, based upon alignment with other vertebrate MBPs, contained six of the seven MBP exons; only exon II was absent. The smaller product encoded a 141 amino acid protein that lacked exon II and exon V. There were 26 and 30 nucleotide differences between Squalus and Heterodontus, and Raja and Heterodontus, respectively. Sequences from Squalus and Raja were far more similar, having only five nucleotide differences. Both isoforms of elasmobranch MBP contain 18.5% basic (lysine plus arginine) amino acids, compared with 17.5% in mammalian MBPs comprised of the corresponding exons. Northern blot analysis of whole brain total RNA revealed a single band of 2.5 kb in Squalus, and three bands of 1.2, 1.4, and 2.3 kb in Raja. The finding that MBPs of a Squalomorph shark and a Batoidea ray are closer to one another than either is to the Galeomorph sharks suggests that MBP sequence information may prove useful in classifying modern day Chondrichthytes. © 1993 Wiley‐Liss, Inc.
Myelin basic protein mRNA translocation in oligodendrocytes is inhibited by astrocytes in vitro
Authors: Amur‐Umarjee S. , Phan T. , Campagnoni A. T.
Published: January 2005

Myelin basic protein (MBP) mRNAs are translocated from cell bodies into the slender processes connecting oligodendrocyte somas with the myelin sheath in vivo. This translocation was observed in mixed glial cultures prepared from newborn mouse brains and it occurred in approximately 25% of the cells expressing the gene. However, when “enriched” oligodendrocytes were prepared by shaking them free of other glial cells, MBP mRNA translocation occurred into the processes of essentially all of the cells. When enriched oligodendrocytes were plated back onto astrocytes, MBP mRNA was observed to be confined to the cell bodies of almost all the cells, indicating a marketd inhibition of translocation of the mRNA. This inhibition of mRNA translocation did not appear to be mediated through soluble factors secreted by astrocytes or by “astromatrix,” but rather through physical contact between the oligodendrocytes and astrocytes. Intact, but not necessarily live, astrocytes were required for the inhibition of mRNA translocation in the oligodendrocytes. Fibroblasts and a neuroblastoma cell line, SKN‐SH, did not inhibit MBP mRNA translocation in oligodendrocytes suggesting that astrocyte surface‐specific components might be involved in the interaction between astrocytes and oligodendrocytes in culture. These results suggest that contact between these two cell types can influence intramolecular events related to myelinogenesis. © 1993 Wiley‐Liss, Inc.
Nerve growth factor stimulates GAP‐43 expression in PC12 cell clones independently of neurite outgrowth
Authors: Burry R. W. , Perrone‐Bizzozero N. I.
Published: January 2005

Expression of the growth associated protein GAP‐43 (B‐50, F1, neuromodulin) increases with the onset of neuronal development as seen by the growth of axons. To investigate the relationship of the signaling events leading to GAP‐43 expression and neurite outgrowth, we examined PC12 clones with different phenotypes. Three clones, PC12‐NO9, PC12‐N15, and PC12‐N21, responded to NGF with increased expression of GAP‐43, but only two clones, PC12‐N15 and PC12‐N21, responded with growth of neurites. Similar increases in expression of GAP‐43 were obtained when these clones were exposed to the phorbol ester PMA. Thus, NGF and PMA induced GAP‐43 expression in PC12‐NO9 cells in the absence of neurite outgrowth. In contrast, all three clones, were able to respond to forskolin (FOR) by initiation of long neurites which had synaptophysin in the growth cones, but showed only low levels of GAP‐43. Combined stimualtion of PC12‐NO9 cells with FOR and PMA both initiated neurites and increased expression of GAP‐43 as seen in normal PC12 clones were also able to respond to FOR with increased neurite outgrowth in the presence of low levels of GAP‐43. The dissociation of GAP‐43 expression and growth of neurites observed in PC12‐NO9 cells suggests that signaling mechanisms can independently regulate GAP‐43 expression and neurite outgrowth during neuronal differentiation. © 1993 Wiley‐Liss, Inc.
Bradykinin and capsaicin stimulate cyclic GMP production in cultured rat dorsal root ganglion neurons via a nitrosyl intermediate
Authors: Bauer M. B. , Simmons M. L. , Murphy S. , Gebhart G. F.
Published: January 2005

Dorsal root ganglion (DRG) neurons express receptors for bradykinin and capsaicin, both algesic substances. Administration of bradykinin or capsaicin to neurons cultured from embryonic rat DRG stimulated the production of cyclic GMP but in nonneuronal DRG cultures. Bradykinin‐evoked Cyclic GMP production was mediated by B2 receptors and was unaltered by indomethacin. Both bradykinin‐ and capsaicin‐stimulated cyclim GMP production required Ca2+ and was inhibited by methylene blue. Furthermore, methylene blue attenuated basal cyclic GMP production in DRG neurons, suggesting tonic cyclic GMP production in these Cells. L‐NG‐monomethyl arginine inhibited both bradykinin‐and capsaicin‐stimulated cylic GMP production as well as basal cyclic GMP production. These findings suggest the involvement of a nitrosyl compound in bradykinin‐and capsaicinstimulated cyclic GMP production and in tonic cyclic GMP production in DRG Neurons. © 1993 Wiley‐Liss, Inc.