bluuebunny/crossref_metadata_2025 huggingface dataset:
Neural influences on synovial mast cell density in rat
Authors: Levine J. D. , Coderre T. J. , Covinsky K. , Basbaum A. I.
Published: January 2005

The mast cell, an immunocompetent cell that contributes to neurogenic inflammation in a variety of tissues, including synovium, is found in close proximity to peripheral terminals of unmyelinated primary afferents and sympathetic postaganglionic nerve terminals. In this study we evaluated the hypothesis that the density of mast cells in synovial tissue is dependent on the neural innervation. In normal rats, we found that the density of mast cells in the ankle joint capsule, which is heavily innervated, is greater than in the capsule of the knee, which is less densely innervated. Selective lesions of unmyelinated primary afferents with capsaicin, or of sympathetic postganglionic neurons with guanethidine, produced a significant decrease in mast cells; surgical removal of the parasympathetic ganglia did not significantly affect mast cell density. Finally, the number of mast cells in the synovial joint of spontaneously hypertensive rats, which have increased sympathetic activity, was significantly greater than in normotensive control rats. These observations support the hypothesis that the innervation in the synovial joint of the rat, by unmyelinated afferents and sympathetic efferents, exerts a trophic effect on mast cell density.
Morphological changes in cultures of hippocampus following prenatal irradiation in the rat
Authors: Hamdorf G. , Shahar A. , Cervós‐Navarro J. , Schemer A. , Sparenberg A. , Skoberla A.
Published: January 2005

The effect of prenatal irradiation was studied in organotypic cultures of hippocampus, prepared from newborn rats that had been exposed to wholebody irradiation of 1 Gy from a 60Co‐source at day 13 of pregnancy. Light and electron microscopic observaions showed remarkable damage to neuronal mitochondria accompanied by extensive swelling, vacuolation of the Golgi complex, and formation of multilamellar bodies and vesicles of the lysosomal type. In contrast to neuronal alterations, no delay in synaptogenesis or onset of myelination was observed based upon the absence of significant morphological changes in synapses and myelin sheaths. Using this tissue culture model it could be confirmed that prenatal exposure to irradiation, even at low doses, induces specific morphological changes in the brain.
Changes on cerebral antioxidant enzymes, peroxidation, and the glutathione system of frogs after aging and catalase inhibition
Authors: de Quiroga G. Barja , Pérez‐Campo R. , López‐Torres M.
Published: January 2005

The five major antioxidant enzymes, glutathione, and in vivo or in vitro stimulated (Fe++‐ascorbate) peroxidation were similar in old and young Rana perezi frogs. Long‐term (2.5 months) treatment with aminotrizole strongly decreased cerebral catalase (CAT) activity and increased in vivo but not in vitro peroxidation in the brain. This suggests that the increase in endogenous brain peroxidation after CAT inhibition is due to an increased free‐radical attack on cerebral membranes, and not to a possible increase in their sensitivity to peroxidative damage. The increase of in vivo peroxidation is especially remarkable taking into account the low levels of CAT present in the vertebrate brain. On the other hand, these changes were not accompanied by any effect on the survival of the animals. Comparison of these results with those obtained in other species suggests the possibiltity that O2‐free radicals be of minor importance in relation to brain aging in animals with low rates of oxygen consumption.
Arachidonec acid metabolism in grebil cerebra: Effects of ischemia
Authors: Hamm T. F. R. , Dorman R. V.
Published: January 2005

Pentobarbital pretreatmint may be used to predict biochemical events involved in ischemic brain damage following bilateral carotid artery ligations in the gerbil, since it reduces the subsequent edema and mortality. The effects of this anesthetec on the ischemia‐induced modifications of cerebral arachidonic acid metabolism were investigated, in order to correlate observed alterations with tissue damage. Cerebral lipids were radiolabeled in vivo with [3H] arachidonec acid prior to 10 min of cerbral ischemia and 0‐120 min of perfusion. Ischemia stimulated a 97.3% increase in unesterified [3H] arachidonate, which was dut to the loss of label from choline, inositol, and ethanolamine glycerophospholipids. Tissue reperfusion stimulated further reductions in [3H] choline and [3H] inositol glycerophospholipids, while ethanolamine glycerophospholipip and trigyceride labeling increased. Inositol glycerophospholipid, but not chline glycerophospholipid, labeling returned to control level by 60 min of reperfusion. Pentobarbital pretreatment reduced the accumulation of [3H] arachidonate by 56.2% during ischemia. It increassed the recovery of [3H]ethanolamine glycerophospholipids during the ischemic period and [3H]choling glycerophospholipids dduring the first 5 min of reperfusion. Thessses effects accountedd for the feduction of unesterifiedd [3H]arachidonate obsserved during ischemia and reperfusion.
West nile virions aligned along myelin lamellae in organotypic spinal cord cultures
Authors: Shahar A. , Lustig S. , Akov Y. , David Y. , Schneider P. , Friedmann A. , Levin R.
Published: January 2005

Organtypic spinal cord cultures infected with West Nile Virus (WNV) exhibited a remarkable arrangement of virions among lamellae of the myelin sheath. Virions were first observed in neurons and only at day 4 after infection appeared within the myelin lamellae. Virions were observed only in the central myelin, aligned along the interperiod lines and therefor attached to the outer side of the oligodendrocyte membrane. In spite of this peculiar location of the virions, their presence was not associated with severe damage to the axon or to the myelin sheath. The causes of the formation of this viral pattern, its morphological features, and the role which it might have in viral infection in vivo are considered.
Regulation of thyroid hormone receptor and c‐erbA mRNA levels by butyrate in neuroblastoma (N2A) and glioma (C6) cells
Authors: Yusta B. , Ortiz‐Caro J. , Bedo G. , Pascual A. , Aranda Ana
Published: January 2005

Butyrate produced a biphasic modulation of the thyroid hormone receptor in neuroblastoma N2A cells increasing receptor number by 20–35% at concentrations 0.25–0.75 mM and decreasing receptor levels by 30–55% at 2–4 mM. The half‐life of the receptor, as assessed by its disappearance after incubation with 18 μM cycloheximide was 8.4 hr in control cells and 10.3 hr and 5.0 hr in cells incubated with 0.25 and 4 mM butyrate, respectively. This compound increased the abundance of multyacetylated forms of histone H4 from 30% in control cells to almost 70% with butyrate 4 mM. In glioma C6 cells, the fatty acid produced a dose‐dependent increase of receptor levels (up to 3–4‐fold with 2–5 mM butyrate) and had little effect in increasing multiacetylation (from 30% in controls to 42–46% with 2–5 mM butyrate). Recent studies have shown that the c‐erbA proto‐oncogen codes for the thyroid hormone receptor. In N2A and C6 cells, 2 c‐erbA‐related mRNAs, one measuring 2.6 kb and the other 6 kb, were detected. Both forms were differently regulated by butyrate. This compound decreased the abundance of the 2.6 kb forms in both cell types, even at the concentrations at which there was an elevation of receptor levels. Only the largest mRNA correlated with receptor concentration increasing by 2–3‐fold after treatment of C6 cells with butyrate, and undergoing a smaller but biphasic change in N2A cells.

Our data suggest that modification of chromatin structure probably secondary to acetylation induces changes in thyroid hormone receptor levels in neuroblastoma and glioma cells by affecting both receptor stability and receptor mRNA levels.
Excitatory amino acid neurotoxicity in cultured retinal neurons: Involvement of N‐methyl‐D‐Aspartate (NMDA) and Non‐NMDA receptors and effect of ganglioside GM1
Authors: Facci L. , Leon A. , Skaper S. D.
Published: January 2005

Cultures of chicken day 8 embryo retinal cells, essentially free of contaminating non‐neuronal elements, were used to examine the neurotoxicity of various excitatory amino acid transmitter receptor agonists. At 7 days in vitro, N‐methyl‐D‐aspartate (NMDA), following 24 hr exposure to 0.1‐1.0 mM, destroyed 60‐70% of the multipolar neurons, but apparently spared photoreceptors. The cytotoxic effect of NMDA was prevented by extracellular Mg2+ or phencyclidine, suggesting a role for the NMDA ion channel; competitive NMDA antagonists were also neuroprotective. The mixed excitatory amino acid receptor agonist glutamate (0.1‐1.0 mM) was also neurotoxic (∼70% loss of multipolar neurons) and strongly blocked by NMDA (but weakly by non‐NMDA) antagonists and Mg2+, indicating a major action at NMDA receptors. As with NMDA, glutamate did not appear to affect photoreceptors. The neurotoxic action of kainate against multipolar retinal neurons, as reported by others, was confirmed here. Kainate neuronal injury was sensitive to the quinoxalinedione non‐NMDA antagonists 6,7‐dinitroquinoxaline‐2,3‐dione (DNQX) and 6‐cyanoquinoxaline‐2,3‐dione (CNQX), but not to Mg2+ or phencyclidine. Ibotenate and quisqualate, even at millimolar concentrations, were not neurotoxic. The monosialoganglioside GM1 was also effective in reducing NMDA and non‐NMDA agonist neurotoxicity to retinal neurons. Maximal ganglioside benefit required 1‐2 hr of pre‐treatment with 100‐200 μM GM1.The percentage of multipolar neurons remaining after the neurotoxin insult approximately doubled with GM1 treatment. Gangliosides may thus have a therapeutic potential in excitatory amino acid‐initiated neuropathologies.
Phosphoinositide breakdown in isolated myelin is stimulated by GTP analogues and calcium
Authors: Golly F. , Larocca J. N. , Ledeen R. W.
Published: January 2005

Purified myelin from rat brainstem, prelabeled in vivo by intracerebral injection of [3H]myoinositol, showed enhanced breakdown of phosphoinositides on treatment with 5'‐guanylylimidodiphosphate [Gpp‐(NH)p] and Ca2+. Concentration variation of the former in the presence of Ca2+ snowed a dose‐dependent release of inositol 1,4‐bisphosphate (IP2) and inositol 1,4,5‐trisphosphate (IP3), while inositol 1‐phosphate (IP) release was erratic. Concentration‐dependent release of IP2 and IP3 was also observed with Ca2+ as the variable in the presence of Gpp(NH)p. Carbachol, when present, did not enhance the stimulatory effect of Gpp(NH)p alone. Addition of diphosphoglycerate during incubation enhanced IP3 at the expense of IP2, suggesting the presence of IP3 phosphatase in mylein.
Type II glucocorticoid receptors are expressed in oligodendrocytes and astrocytes
Authors: Vielkind U. , Walencewicz A. , Levine J. M. , Bohn Martha Churchill
Published: January 2005

Glucocorticoid hormones affect gene expression directly at the level of transcription via intracellular receptors that translocate to the nucleus in the presence of steroid. In the brain, two types of high‐affinity receptors bind glucocorticoids, the type I, mineralocorticoid receptor and the type II, glucocorticoid receptor (GR). Both receptor types are expressed by many types of neurons. Although binding studies have suggested that glial cells may also express receptors, the expression of these receptors in specific classes of glia has not been studied previously. This immunocytochemical study was undertaken to determine which of the different classes of glial cells express type II GR. Primary cultures of mixed glial cells from rat cerebrum and cerebellum, purified oligodendrocytes and astrocytes. as well as two glial tumor cell lines were screened for the expression of glucocorticoid receptors using a mouse monoclonal antibody directed against rat liver GR (BuGR‐2). Glial cell types were identified by morphology and immunoreactivity (IR) with antibodies directed against glial fibrillary acidic protein (GFAP), cyclic nucleotide phosphodiesterase (CNP), or myelin basic protein (MBP). Double immunofluorescence microscopy revealed that all GFAP‐IR cells (type 1 and type 2 astrocytes), all CNP‐ or MBP‐IR cells (oligodendrocytes), as well as immature and intermediate cell types expressed GR, although at different levels. C6 glioma and JScl1 Schwannoma cells were observed to express moderate to high levels of GR. Furthermore, cells grown in the absence of glucocorticoids had diffuse GR staining over the cytoplasm, whereas cells grown in the presence of the synthetic glucocorticoid dexamethasone had strong nuclear staining. These results demonstrate that, in vitro, all classes of glial cells express glucocorticoid receptors that can translocate to the nucleus in the presence of hormone. These observations suggest that glial cells are major targets for glucocorticoid‐directed control of gene transcription in the nervous system.
Laminin‐mediated process formation in neuronal cells involves protein dephosphorylation
Authors: Weeks B. S. , DiSalvo J. , Kleinman H. K.
Published: January 2005

Laminin mediates neural adhesion and process formation. A possible signal transduction pathway for laminin was investigated in both NG108‐15 and PC12 neuronal cells using radiolabeling studies as well as various stimulators and inhibitors of phosphatases and kinases. Using [32p]‐ortho‐phosphate, laminin caused a decrease in the TCA‐precipitable counts. Further, laminin stimulated dephosphorylation of laminin binding proteins of 110 kDa, 67 kDa, and 45 kDa and this dephosphorylation was blocked by the phosphatase inhibitor, okadaic acid, and the protein kinase C stimulator, TPA. The phosphatase inhibitors okadaic acid and vanadate, as well as the protein kinase C stimulators, TPA and DAG, blocked laminin‐mediated process formation. Inhibitors of kinase activity such as H‐7, H‐8, and H‐9 increased laminin‐mediated neural process formation. Since phosphate incorporation into laminin‐binding proteins is decreased by laminin and because both phosphatase inhibitors and kinase stimulators inhibit laminin‐mediated process formation, we conclude that dephosphorylation events promote the neural cell response to laminin.
Aspartate‐70 to glycine substitution confers resistance to naturally occurring and synthetic anionic‐site ligands on inovo produced human butyrylcholinesterase
Authors: Neville L. F. , Gnatt A. , Loewenstein Y. , Soreq H.
Published: January 2005

The “atypical” allelic variant of human butyrylcholinesterase (BuChE) can be characterized by its failure to bind the local anesthetic dibucaine, the muscle relaxant succinylcholine, and the naturally occurring steroidal alkaloid solanidine, all assumed to bind to the charged anionic site component within the normal BuChE enzyme. A single nuclcotide substitution conferring a change of aspartate‐70 into glycine was recently reported in the CHE gene encoding BuChE from several individuals having the “atypical” BuChE phenotype, whereas in two other DNA samples, this mutation appeared together with a second alteration conferring a change of serine‐425 into proline. To separately assess the contribution of each of these mutations toward anionic site interactions in BuChE, three transcription constructs were engineered with each of these substitutions alone or both of them together. Xenopus oocyte microinjection of normal or mutated synthetic BuChEmRNA transcripts was employed in conjunction with biochemical analyzes of theresultant recombinant BuChE variants. The presence of the Gly‐70 mutation alone was found to render the enzyme resistant to 100 μM solanidine and 5 mM succinylcholine; concentrations sufficient to inhibit the “normal”, Asp‐70 containing BuChE by over 50%. Furthermore, when completely inhibited by the organophosphorous poison diisopropylfluorophosphate (DFP), Gly‐70 BuChE failed to be reactivated by 10 mM of the cholinesterase‐specific oxime pyridine 2‐aldoxime methiodide (2‐PAM); a concentration restoring about 50% of activity in the “normal” Asp‐70 recombinant enzyme. The Pro‐425 mutation alone had no apparent influence on BuChE interactions with any of these ligands. However, it conferred synergistic effects on some of the anionic site changes induced by the Gly‐70 mutation. Thus, in addition to being resistant to soianidine and to 2‐PAM reactivation, the Gly‐70/Pro‐25 variant completely failed to bind either dibucaine (1 mM) or succinylcholine (1,000 mM), 20‐fold higher concentrations than those inhibiting the single Gly‐70 mutant. In view of the evolutionary conserved Asp‐70 and Ser‐425 peptide domains in all known cholinesterases, these findings suggest that the Asp‐70 residue serves as part of the anionic site responsible for binding charged substrates throughout the family of cholinesterases and that genetically linked mutations in these two peptide domains may confer selection advantage through their synergistic effects on BuChE resistance to inhibitors.
Somatic cell genetic analysis of the galactocerebrosidase gene: Lack of complementation in human Krabbe disease/twitcher mouse cell hybrids
Authors: Skiba M. C. , Lyerla T. A. , Konola J. T. , Raghavan S.
Published: January 2005

The inherited deficiency of galactosylceramide β‐galactosidase (E.C. 3.2.1.46: galactocerebrosidase) activity results in globoid cell leukodystrophy in humans (Krabbe disease) and in mice (twitcher mutant). To determine whether Krabbe patients, cells complement twitcher cells to produce, in hybrid combination, greater than deficient levels of galacto‐cerebrosidase activity, five separate crosses were made between an established twitcher mouse cell line and five cell strains from unrelated Krabbe disease patients. A total of 57 twitcher mouse/Krabbe somatic cell hybrid lines developed from all of these crosses were deficient in galactocerebrosidase activity despite the presence of human chromosomes 14 or 17, which have been previously implicated as bearing the galactocerebrosidase gene. A control cross between twitcher mouse/positive control human fibroblasts resulted in 14 of 21 independent hybrid lines that expressed higher than deficient levels of galactocere brosidase activity. The lack of complementation between Krabbe disease patient and twitcher mutant mouse cells provides further evidence that the twitcher mouse is an authentic murine model for Krabbe disease and supports the hypothesis that the mutations in both species are within the structural gene for the galactocerebrosidase enzyme.
Expression of a chimeric VIP gene is targeted to the intestine in transgenic mice
Authors: Agoston D. V. , Bravo D. T. , Waschek J. A.
Published: January 2005

We showed previously that a gene construction that consisted of 5.2 kb of 5' flanking sequence, the first exon, and part of the first intron of the human gene encoding vasoactive intestinal peptide (VIP) fused to the reporter gene chloramphenicol acetyltransferase (CAT) fully mimicked the diverse behavior of the endogenous VIP gene when transfected into subclones of the human neuroblastoma cell line SK‐N‐SH (Waschek et al., 1988). To determine if the same sequences were sufficient to target expression of a reporter to VIP‐producing tissues in the mouse, we initiated a pilot study in which we generated four transgenic mice or mouse lines that contained the VIPCAT fusion gene. Detectable levels of CAT were found in the ileum of either founder or offspring of each of the transgenic mouse lines. In all other tissues tested, CAT activity was either below the level of detection or the transgene was not expressed, with the exception of one mouse in which ectopic expression in the cerebellum was observed. The results indicate that the VIP sequences utilized were sufficient to direct expression of the transgene to the intestine, but not necessarily to other sites of VIP expression.

To investigate what specific DNA sequences might confer VIP expression in the intestine and other sites, we analyzed further the VIP gene in SK‐N‐SH subclones using VIP/luciferase fusion gene constructions. A 0.6 kb DNA fragment located between 4.0 kb and 4.6 kb upstream from the VIP transcriptional start site was found to impart a high level of expression in one subclone and an increased degree of phorbol ester induction in another. These and other data indicate that multiple transcriptional elements control VIP expression in neuroblastoma cells and are candidates as mediators of VIP gene expression in the intact animal.
Quisqualate‐ and NMDA‐sensitive [3H]glutamate binding in primate brain
Authors: Young A. B. , Dauth G. W. , Hollingsworth Z. , Penney J. B. , Kaatz K. , Gilman S.
Published: January 2005

Excitatory amino acids (EAA) such as glutamate and aspartate are probably the neurotransmitters of a majority of mammalian neurons. Only a few previous studies have been concerned with the distribution of the subtypes of EAA receptor binding in the primate brain. We examined NMDA‐ and quisqualate‐sensitive [3H]glutamate binding using quantitative autoradiography in monkey brain (Macaca fascicularis). The two types of binding were differentially distributed. NMDA‐sensitive binding was most dense in dentate gyrus of hippocampus, stratum pyramidale of hippocampus, and outer layers of cerebral cortex. Quisqualate‐sensitive binding was most dense in dentate gyrus of hippocampus, inner and outer layers of cerebral cortex, and molecular layer of cerebellum. In caudate nucleus and putamen, quisqualate‐ and NMDA‐sensitive binding sites were nearly equal in density. However, in globus pallidus, substantia nigra, and subthalamic nucleus, quisqualate‐sensitive binding was several‐fold greater than NMDA‐sensitive binding. In thalamus, [3H]glutamate binding was generally low for both subtypes of binding except for the anterior ventral, lateral dorsal, and pulvinar nuclei. In the brainstem, low levels of binding were found, and strikingly the red nucleus and pons, which are thought to receive glutamatergic projections, had approximately 1/20 the binding observed in cerebral cortex.

These results demonstrate that NMDA‐ and quisqualate‐sensitive [3H]glutamate binding are observed in all regions of primate brain, but that in some regions one subtype predominates over the other. In addition, certain areas thought to receive glutamatergic projections have low levels of both types of binding.
Passage of MHPG from plasma to CSF in a non‐human primate
Authors: Faull K. F. , Pascoe N. , Maddaluno J. , Greene K. A. , Wiener S. G.
Published: January 2005

In experimental protocols with humans and non‐human primates, cerebrospinal fluid (CSF) concentrations of 3‐methoxy‐4‐hydroxyphenylethylene glycol (MHPG), the predominant end‐product of norepinephrine metabolism in the mammalian central nervous system (CNS), have been widely used as an index of the rate of CNS norepinephrine metabolism. However, an earlier investigation showed that there was slow but free exchange between plasma and CSF MHPG. To define more precisely the time‐course of equilibration of plasma and CSF MHPG, we intravenously administered 100 μg/kg of [2H3]‐MHPG to drug‐naive squirrel monkeys. Measurements were made of the concentrations of [2H3]‐ and [1H]‐MHPG in plasma and cervical CSF samples collected at time points from 10 min to 4 hr thereafter. The results indicated that neither plasma nor CSF concentrations of [1H]‐MHPG changed during the course of the experiment, and that [2H3]‐MHPG appeared in the CSF within 10 min of intravenous administration. The maximal plasma and CSF concentrations of [2H3]‐MHPG were 7.6‐ and 2.3‐fold higher than the respective concentrations of [1H]‐MHPG. The plasma and CSF pools of [2H3]‐MHPG reached concentration equilibrium within 30 min, and thereafter the temporal decline in concentration of [2H3]‐MHPG was the same in plasma and CSF. These results demonstrate that MHPG rapidly crosses from plasma to CSF, and support the suggestion that this factor be included in any attempts to estimate norepinephrine turnover in the CNS from measurements of steady‐state MHPG concentrations in CSF or plasma.
Correlations of regional postmortem enzyme activities with premortem local glucose metabolic rates in alzheimer's disease
Authors: McGeer E. G. , McGeer P. L. , Harrop R. , Akiyama H. , Kamo H.
Published: January 2005

Correlations were sought between local cerebral metabolic rates (LCMRs) for glucose in various regions of the cortex, determined in premortem PET scans, with the regional activities of choline acetyltransferase (ChAT), acetylcholinesterase (AChE), β‐glucuronidase (Gluc, a probable index of reactive gliosis), and phosphate‐activated glutaminase (PAG, a possible indice of the large pyramidal neurons) measured on postmortem tissue. Significant negative correlations between LCMRs and Gluc activities were found in 6 PET‐scanned cases of Alzheimer disease (AD), and positive correlations of LCMRs with PAG were found in 5. By contrast, a positive correlation with ChAT and AChE was found in only 1. The results are consistent with the metabolic deficits in AD being primarily a reflection of local neuronal loss and gliosis. Similar data on two cases of Huntington's disease showed no significant correlations, while 1 patient with Parkinson dementia showed a significant (negative) correlation only with Gluc.
Localization of NGF receptors in normal and Alzheimer's basal forebrain with monoclonal antibodies against the truncated form of the receptor
Authors: Loy R. , Heyer D. , Clagett‐Dame M. , DiStefano P. S.
Published: January 2005

Four new monoclonal antibodies to the extracellular domain of the nerve growth factor receptor (NGFR) have been evaluated for their specificity to NGFR and their utility in localizing NGFR in human brain. All four antibodies, as well as Me20.4, show similar cellular localization and patterns of immunoreactivity in basal forebrain neurons. NGFR monoclonal antibody XIF1 stains optimally over the widest range of concentrations, with staining being reduced only slightly at less than 10 pg/ml or more than 100 ng/ml, and produces the lowest background of those tested. Staining with all NGFR monoclonal antibodies is blocked by the addition of as little as 5‐fold excess human recombinant truncated NGFR protein. The distribution of NGFR‐containing neurons is similar to that previously described in normal human fore‐brain, as is the reduction in cell size in nucleus basalis (Ch4am) in brains from patients with Alzheimer's disease. In addition, we find evidence in the two Alzheimer's cases examined for a previously unreported loss of cells in the horizontal limb nucleus of the diagonal band (Ch3) in Alzheimer's disease. The loss of these neurons, which in normal brain have characteristic varicose dendritic processes extending to the pial surface adjacent to the cisternal space, may indicate a change in the relationship of NGF‐sensitive neurons to the vasculature. Since these neurons project to olfactory bulb and cortex in rodent and primate brains, their loss may also reflect damage to the olfactory system in Alzheimer's disease.
Neurobiology of human oligodendrocytes in culture
Authors: Kim S. U.
Published: January 2005

Enriched populations of oligodendrocytes were isolated from adult human brains of 3‐15 hours postmortem using the trysinin digestion‐Percoll density gradient method and were cultured for an extended period of time up to 6 months. Cell type specific antigens that were expressed by oligodendrocytes were galactocerebroside, myelin basic protein, proteolipid protein, 2'3'‐cyclic nucleotide 3'‐phosphehydrolase and myelin‐associated glycoprotein. In addition, HLA‐A, B, C and HLA‐DR, respectively, Class I and Class II antigens of the major histocompatibility complex, were demonstrated on oligodendrocytes. Three classes of gangliosides, GM1, GM4, and GD3, were also demonstrated on oligodendrocytes, while GM1 and GM4 gangliosides were detected on the surface of astrocytes. The presence of “transitional” or “bipotential” glial cells that were derived from oligodendrocytes and that expressed both oligodendroglial and astrocytic phenotypes was demonstrated. Treatment of the cells by cyclic AMP and its derivatives reversed this dual phenotypic expression back to the oligodendroglial trait. Electron microscopic examination of oligodendrocytes indicated that they were capable of synthesizing and assembling myeiin sheaths in culture in the absence of any neuronal signal input.
Distribution of proteolipid protein and myelin basic protein in cultured mouse oligodendrocytes: Primary vs. secondary cultures
Authors: Konola J. T. , Tyler B. M. , Yamamura T. , Lees M. B.
Published: January 2005

The distribution of proteolipid protein (PLP) and myelin basic protein (MBP) was examined in differentiating Oligodendrocytes of primary and secondary mouse brain cell cultures by single‐ and double‐label indirect immunofluorescence. In primary cultures, MBP and PLP were differentially located in Oligodendrocytes. MBP became concentrated as fine punctate dots lining the edges of processes and as coarse grains in flattened sheet‐like structures. PLP was distributed diffusely throughout cell bodies and processes but was limited to the perimeter of sheets and some processes within sheets. To compare the detailed distribution of PLP and MBP in the absence of underlying cells, a simple method for the growth of isolated Oligodendrocytes in secondary cultures was developed. Cells were maintained in primary culture for 39‐41 days, harvested by scraping, enriched for Oligodendrocytes, and plated at low cell density. After 1 week, isolated Oligodendrocytes had developed long processes and large flattened membranous sheets. MBP and PLP were differentially localized in these cell structures. The sheets contained fine‐grained patches of MBP, which were surrounded by networks of MBP− processes. In contrast, PLP was initially seen throughout the cell bodies and processes. In older cultures, PLP became strikingly concentrated in curvilinear membranous profiles. The observations show that PLP and MBP aie differentially located in cultured mouse Oligodendrocytes. Further‐more, the precise distribution of these myelin‐specific antigens is dependent on culture conditions.
Beta amyloid precursor protein mediates neuronal cell‐cell and cell‐surface adhesion
Authors: Breen K. C. , Bruce M. , Anderton B. H.
Published: January 2005

The beta‐amyloid precursor protein (APP) is a membrane‐bound glycoprotein which has been proposed to play a role both as a growth factor and a mediator of cell adhesion. Using the Ncuro‐2A neuroblastonia cell line, we have investigated the capacity of APP to mediate neural cell adhesion. The cells express the protein at a high level, the immunohistochemical staining pattern at the level of the membrane having a punctate pattern. Fab' fragments of antibodies to the extracellular portion of the molecule were found to inhibit cell binding to a collagen substrate, but not to laminin, fibronectin, or poly‐I‐lysine. Fab' fragments of antibodies to the nerve cell adhesion molecule N‐CAM also inhibited binding of Neuro‐2A cells specifically to collagen. This inhibition of cell‐surface binding was accompanied by a repression of neurite outgrowth in differentiating cells in the presence of antibodies. APP antibodies also inhibited neuron‐neuron and neuron‐glial binding, but not glial‐glial cell adhesion. These data suggest that the APP, which is expressed primarily on differentiated neuronal cells, may play a role in the mediation of both cell‐cell and cell‐substrate adhesion.
Identification of a cis‐acting positive regulatory element of glial fibrillary acidic protein gene
Authors: Sarid J.
Published: January 2005

Developmental regulation of astrocyte‐specific expression of the glial fibrillary acidic protein (GFAP) gene reflects transition of immature glioblasts to mature astrocytes. Described here is the cloning and sequencing of the 5′‐flanking region of the mouse GFAP gene. It contains a glial‐specific positive cis‐acting regulatory element that directs preferential expression of a linked reporter gene when transfected into GFAP‐positive glioblasioma cells. Sequence analysis of this region revealed the presence of a putative AP‐1 binding site, implying a possible role for AP‐1 factors in the astroglial‐specific expression of the GFAP gene.
Excitatory amino acid‐induced alterations of cytoplasmic free Ca2+ individual cerebellar granule neurons: Role in neurotoxicity
Authors: Milani D. , Guidolin D. , Facci L. , Pozzan T. , Buso M. , Leon A. , Skaper S. D.
Published: January 2005

The effects of glutamate on intracellular free Ca2+ [Ca2+]i, and neurotoxicity were compared in cerebellar granule neurons in vitro. [Ca2+]i, was measured with fura‐2 and digital fluorescence imaging microscopy; neurotoxicity was monitored using a vital dye and colorimetric analysis. Glutamate produced dosedependent increases in [Ca2+]XSi transient for glutamate concentrations in a range of : 0.01–0.5 μM and sustained for higher levels of glutamate. The ED50 for the [Ca2+]i response to glutamate was. 6 μM. The LQ50 for glutamate‐induced neurotoxicity was similar, i.e., 10 μM The effect of glutamate on [Ca2+]i was greatly dmiinished when external Ca2+ was removed and blocked by Mg2++ N‐methyl‐D‐aspartate (NMDA)‐type receptor antagonists. The latter conditions as well as preloading granule neurons with the intracellular Ca2+ chelator quin2 largely prevented glutamate cytotoxicity. The neurotoxic effect of glutamate required incubations with the stimulus for 10–20 min at 25°C. Withdrawal of glutamate after this period was accompanied by a prolonged alteration in [Ca2+]i. Pretreatment cells with the ganglioside GM1 reduced this late increase in [Ca2+]i, as well as the neurotoxic effects of glutamate. This indicates that glutamate‐induced neurotoxicity results from a composite of diverse temporal alterations in Ca2+ homeostasis and that blunting any of these components reduces excitotoxicity.
Platelet‐activating factor increases glutamine synthetase activity in early and late passage C‐6 glioma cells
Authors: Kentroti S. , Baker R. , Lee K. , Bruce C. , Vernadakis A.
Published: January 2005

Previous studies from this laboratory have shown that C‐6 rat glioma cells (2B clone) exhibit specific phenotypic characteristics depending on passage in culture and that these populations respond differentially to addition of various exogenous compounds to the medium. Early passage (<25) C‐6 glial cells express low glutamine synthetase activity (a marker for astrocytes) and with increasing cell passage (>70) C‐6 glial cells express more astrocytic properties with respect to both glutamine synthetase (GS) and morphology. In this study, cells from both early (glioblastic) and late (astrocytic) passage were examined for their response to the phospholipid, platelet‐activating factor (PAF). We found that PAF increased GS activity in early passage (glioblastic) cells and more importantly it increased GS activity in late passage cells, already committed to the astrocytic pheriotype. Furthermore, cells from both passages failed to respond to addition of lyso‐PAF, the non‐biologically active analog of PAF, to the medium, By following the uptake of 3H‐PAF into cells, we observed that >90% of the phospholipid was taken into the cells within the first hour of incubation. We compared the PAF effects with that of dibutyryl cyclic AMP (dBcAMP) and RO20‐1724, a phosphodiesterase inhibitor. Cells from the early passage responded to both dBcAMP and RO20‐1724 treatments with a significant increase in GS activity whereas cells from the late passage showed no significant change, confirming earlier reports from this laboratory. These findings indicate that the response of C‐6 glioma cells to PAF (at least in the late passage) is not mediated via cyclic AMP. We suggest that in early passage cells PAF promotes expression of the astrocytic phenotype and in late passage cells PAF mediates a gliosis‐type response.
Oligodendroglial cell death induced by oxygen radicals and its protection by catalase
Authors: Kim Y. S. , Kim Seung U.
Published: January 2005

The cytotoxic effects of oxygen radicals have been studied in enriched population of mature bovine oligodendrocytes in culture. Oxygen radicals were generated enzymatically by glucose and glucose oxidase, and hypoxanthine and xanthine oxidase combinations. Cytotoxicity was assessed by trypan blue exclusion and percentage lactate dehydrogenase release into the culture media. Incubation of bovine oligodendrocytes with these oxygen radical‐generating systems for 4 hr resulted in significant cell death, especially in the glucose oxidase system. The oligodendrocytes were completely protected by catalase from the cytotoxic effects of both oxygen radical generating systems. However, superoxide dismutase, dimethylsulfoxide and antioxidants such as vitamin E and glutathione did not protect oligodendrocytes from the oxidant‐mediated cytotoxicity. It appears that hydrogen peroxide produced in these oxygen radical‐generating systems gives rise to toxic radicals that induce the cell death of bovine oligodendrocytes in culture.
Developmental and regional expression of choline acetyltransferase mRNA in the rat central nervous system
Authors: Ibáñez Carlos F. , Ernfors P. , Persson H.
Published: January 2005

The developmental and regional expression of choline acetyltransferase (ChAT) mRNA was examined in the rat brain and spinal cord by northern blot analysis and in situ hybridization. ChAT mRNA expression in the brain showed a biphasic increase during development, with a first peak at two weeks postnatally, a marked decrease by the third week, and a second increase between the third and fifth week after birth, indicating that emergence of the cholinergic phenotype occurs at different times in different brain regions. In the spinal cord, ChAT mRNA was detected at similar levels from embryonic stage 13 (E13) until birth, increasing thereafter until adulthood. In the adult rat central nervous system, high levels of ChAT mRNA were detected in the spinal cord and brain stem structures. Lower levels were seen in midbrain, septum, striatum, thalamus, and olfactory bulb. ChAT mRNA containing cells were identified by in situ hybridization in the olfactory tubercule, piriform cortex, striatum, several basal forebrain nuclei, and spinal cord. A nearly twohyphen;fold increase in adult spinal cord ChAT mRNA levels were seen one week after a bilateral crush lesion of the sciatic nerve, indicating that ChAT mRNA expression is regulated during mooneuron regeneration.
Effects of sphinigosine on phorbol ester‐mediated changes in astrocyte morphology and protein phosphorylation
Authors: Hamson Beth C. , Staskavage D. L. , Mobley Philip L.
Published: January 2005

Previous studies indicate that phorbol myristate acetate (PMA) can induce morphological changes in astrocytes cultured from the rat neocortex. PMA also increased 32 P incorporation into several proteins, including glial fibrillary acidic protein (GFAP), vimentin, and proteins with molecular weights of 80,000 (pI 4.5), 50,000 (pI 4.9), and 30,000 (pI 5.5). The present studies were conducted to determine if the morphological effect and the phosphorylation effect of PMA could be blocked by treatment with sphingosine, a protein kinase C inhibitor. Treatment with 15 μM sphingosine inhibited the effect of PMA on astrocyte morphology. This agent also inhibited the increase in phosphorylation mediated by PMA. The percent inhibition ranged from approximately 20% for the 30,000‐M protein to 70% for GFAP. Analysis of phosphorylation sites on GFAP and vimentin using two‐dimensional tryptic mapping techniques indicate that the partial inhibition of phosphorylation is likely the consequence of partial inhibition of protein kinase C rather than a selective inhibition at some phosphorylation sites and not others. In addition to increasing 32P incorporation into various proteins, PMA alsodecreased 32P incorporation in several 20,000‐Mr proteins (pI values of 6.7, 6.4, 6.2, 4.9). However, this effect was not blocked by treatment with sphingosine. This suggests that the actions of PMA to increase and decrease 32P incorporation are mediated by different mechanisms.
Early alterations of Gi/Go protein‐dependent transductional processes in the retina of diabetic animals
Authors: Abbracchio Maria P. , Cattabeni F. , Giulio A. M. Di , Finco C. , Paoletti A. M. , Tenconi B. , Gorio A.
Published: January 2005

The early alterations of G‐protein‐dependent transductional mechanisms have been characterized in the retina of alloxan‐treated diabetic rats. Five weeks after alloxan injection, pertussis toxin radiolabeling of Gi/Go proteins was markedly reduced in the retina of diabetic animals, suggesting either a reduced expression and/or the presence of some structural modification of these G‐protein subtypes. The functional activity of Gs proteins, measured as stimulation of membrane adenylate cyclase by dopamine, did not seem to be impaired at this stage of the pathology; basal adenylate cyclase activity was indeed increased in diabetic rats, consistent with the observed reduction of Gi/Go inhibitory proteins. Such functional alterations of the cAMP producing system were causally related to diabetes induction, since they were reversed by treatment of diabetic animals with insulin. These results suggest that G‐protein dependent transduction mechanisms are altered in the retina of diabetic animals, and that a defect of Gi/Go proteins could represent an early transductional damage in the development of diabetic retinopathy.
Acetylcholinesterase distribution in subpopulations of murine thymocyte
Authors: Rossi Adelaide , Vicini E. , Scarsella G. , Biagioni S.
Published: January 2005

The presence of acetylcholinesterase has been detected in the thymus of several species both biochemically and histochemically.

In this study we have investigated the molecular forms and the level of this enzyme in separate compartments of the murine thymus and in different thymocyte subpopulations.

Similar levels of acetylcholinesterase activity are present both in thymocytes and in the stromal component. Sucrose density gradient analysis revealed the presence of a single molecular form of about 5 S, presumably a dimeric form.

Moreover the results demonstrate a preferential association of AChE with mature thymocyte subsets (Peanut Agglutinin negative and Corticoresistant).

This finding correlates with the preferential sensitivity of these cells to cholinergic drugs and supports the hypothesis that acetylcholinesterase modulates the cholinergic effects on thymocytes.
Phosphyorylation of brain (NA+,K+) ‐ATPase alpha catalytic subunits in normal and epileptic cerebral cortex: I. The audiogenic mice and the cat with a freeze lesion
Authors: Guillaume D. , Grisar T. , Delgado‐escueta A. V. , Bureau‐heeren M. , Laschet J.
Published: January 2005

Partially purified (Na+k+) ‐ATPase (E.C. 3.6.1.3)was investigated in the epileptic cortex of audiogenic DBA/2 mice and in the primary and secondary foci of cats with acute of chronic freeze lesions. No difference in specific activities measured at 3 mM K+ were observed between elliptic and control cortex, except an increase of enzymic activities in the primary focus of acutely lesioned cats. The (Na+, K+) ATPase capalytic subunits were resolved by SDS‐gel electrophoresis and their phosphorylation levels were measured in presence of K+ ions and phenytoin.

K+ was more effective in including maximal de phosphorylation of (Na+, K+)‐Atpase in C57/BL, with identical affinity in the two strains. Phenytoin decreased the net phosphorylation level of (Na+, K+)‐ATPase by about 50% in C57/bl mice, but only by 20% in DBA/2 mice. Both K+ and phenytoin dephosphorylating influence were decreased in primary and secondary foci of acutely lesioned cats. Those changes were limited to the alpha(–) subunit. In chronic cats, the dephosphorylating step of the (Na+, K+)‐ATPase catalytic subnit recovered a normal affinity to K+, but its sensitivity to phenytoin remained decreased.

Those differences in K+ and phenytion influences in brain (Na+, K+)‐ATPase between control and epileptic cortex might be responsible for the ictal transformation and seizure spread. In cats, the alteration of the alpha(–)isoform could mainly affect the glial cells.
Developmental modulation of physicochemical variants of the tailed asymmetric (16S) acetylcholinesterase by neuromuscular activity and innervation in the mouse embryo
Authors: Houenou L. J. , Garcia L. , Verdière‐Sahuqué M. , Villageois A. P. , Rieger F.
Published: January 2005

We have studied the physicochemical properties of acetylcholinesterase (AChE) during embryonic development of normal and functionally impaired mouse skeletal muscle, focusing on the tailed asymmetric (16S) form of the enzyme. The muscle‐specific 16S AChE exists in two different variants. One is associated with extracellular matrix and is high‐salt soluble (HSS, also termed hydrophilic AChE), whereas the other form is anchored to cell membranes and is detergent extractable (DE, or hydrophobic AChE). Before innervation during normal embryonic development, both hydrophilic and hydrophobic 16S AChE exist in equal amounts. After muscle innervation, there was an increase (amounting three‐fold on E18) in the levels of hydrophilic vs. hydrophobic 16S AChE. This alteration of the relative proportions of the two variants of 16S AChE did not occur in chronically inactive muscles either from the mouse mutant, muscular dysgenesis, or from tetrodotoxin‐treated mouse embryos. Taken together with previous reports, the present results suggest that postsynaptic membrane depolarization‐induced Ca2+ important in modulating not only the synthesis of 16S AChE, but also the relative proportions of both physicochemical variants of this molecular form of AChE.
Human monoclonal antineurofilament antibody cross‐reacts with a neuronal surface protein
Authors: Sadiq Saud A. , van den Berg L. H. , Thomas F. P. , Kilidireas K. , Hays A. P. , Latov N.
Published: January 2005

Increased titres of anti‐neurofilament antibodies have been reported in neurodegenerative disorders, and it has been suggested that such antibodies might be pathogenic. We investigated the specificity of an IgA monoclonal antibody (MAb) from a patient with amyotrophic lateral sclerosis which reacted with neurofilaments and bound to the surface of neuroblastoma cells. In Western blots, the immunoaffinity‐purified IgA bound to the 220–kD, high‐molecular‐weight neurofilament protein (NFH) and cross‐reacted with several closely migrating protein bands with apparent mobility of 62–68 kD in neuroblastoma cs and extracts of normal human spinal cord. Following crosslinking to the surface of radiolabeled neuroblastoma cells, the IgA MAb immunoprecipitated a 65–kD protein, indicating that the protein was present on the cell surface and available to the antibodies for binding. Several other MAbs to NFH did not immunostain the surface of neuroblastoma cells or bind to the 65‐kD protein, indicating that the protein was not a fragment of NFH. Thus, antibody binding to the 65–kD protein, possibly by cross‐reacting with NFH, may have contributed to the neuronal degeneration.