bluuebunny/crossref_metadata_2025_split huggingface dataset:
Hypoxic injury to oligodendrocytes: Reversible inhibition of ATP‐dependent transport of ceramide from the endoplasmic reticulum to the golgi
Authors: Kendler A. , Dawson G. 
Published: May 2005

AbstractPrevious studies have shown that gradual progressive hypoxia specifically inhibits the synthesis of the major myelin lipid galactosylceramide (GalCer) in cultured neonatal rat Oligodendrocytes (OLG) (Kendler and Dawson, J Biol Chem 265:12259–12266, 1990). The inhibition of de novo synthesized GalCer (measured by [3H]palmitate incorporation) was accompanied by an increase in the [3H]labeled pool of nonhydroxy fatty acid ceramide, the precursor of GalCer. The decreased galactosylation of NFACer was not due to an inhibition of UDP‐Gal:ceramide:galactosyltransferase activity or to a depletion in available UDP‐Gal. Analysis of subcellular fractionations of OLG membranes on Percoll gradients indicated that NFA ceramide was accumulating in the endoplasmic reticulum (ER) during hypoxia, suggesting that the transport of NFACer from its site of synthesis (ER) to its site of galactosylation, presumably the Golgi, was blocked by hypoxia. This accumulation of ceramide was replicated by lowering ATP levels to 80–90% of control by treating OLG with 12 nM oligomycin, and was reversed by reoxygenation of. the cells. Conversion of [3H] palmitate‐labeled NFACer to GalCer in semi‐intact OLG required both exogenous UDP‐Gal 1 and ATP, further suggesting that the transport of NFACer from the ER to its site of synthesis (cis‐ Golgi) is an energy‐dependent step that is highly susceptible to relatively minor ATP depletion associated with early hypoxic injury. Our results further suggest that ceramide appears to be a good marker for ER eland GalCer is a good marker for the cis‐Golgi.
Synergistic effects of nerve growth factor and phorbol 12‐myristate 13‐acetate on rapid motility and process formation in PC12 cells: The role of laminin
Authors: Glowacka D. , Ginty D. D. , Wagner J. A. 
Published: May 2005

AbstractA126‐1B2 cells, a PKA (cAMP‐dependent protein kinase)‐deficient variant of PC12 cells, but not parental PC12 cells, form processes within 15–30 min of exposure to both nerve growth factor (NGF) and activators of protein kinase C when grown on tissue culture plastic (Glowacka and Wagner, J Neurosci Res 25: 453–462, 1990). Time‐lapse microscopy has demonstrated that these processes are formed by a novel mechanism, i.e., rapid movement of the cell body away from a point of attachment, which morphologically resembles a growth cone. These “fast” neurites are attached to the substratum at a number of points, which display membrane activity in the form of active ruffling and the extension of filopodia and membrane pleats. Thus, these processes are formed by a mechanism distinct from that used by PC12 and other neuronal cells to form processes in culture. Wild‐type PC12 cells also migrate and form fast neurites in response to a combination of NGF and phorbol 12‐myristate 13‐acetate (PMA), when they are grown in conditioned media or plates, suggesting that a secreted factor that can bind to the substratum is essential for the rapid formation of these neurites. Similarly, wild‐type PC12 cells grown on a laminincoated substratum also migrate and form “fast neurites” in response to a combination of NGF and PMA. This rapid migration is attenuated by an antiα1,β1‐integrin antisera, implicating a laminin‐integrin interaction; and it is inhibited by α‐lactalbumin, suggesting an involvement of a β1,4 galactosyltransferase in the response. The formation of fast neurites is not dependent on concurrent protein synthesis, but it is inhibited by lithium, cytochalasin D, and methylthioadenosine or pretreatment of cells with NGF. Thus PC12 cells grown on the appropriate substrate have the ability to migrate rapidly and thereby form neuron‐like processes within minutes of exposure to NGF and PMA. This morphological response to a combination of agents may provide an alternative means by which nerve cells form connections. Alternatively, it may reflect a mechanism that facilitates cellular migration during developmental processes.
Three‐dimensional reconstruction of bovine intradural spinal root myelin by electron microscope tomography
Authors: Sędzik J. , Öfverstedt L.‐G. , Skoglund U. 
Published: May 2005

AbstractElectron microscope tomography was used to reconstruct three‐dimensionally the configuration of heavy metal staining in bovine intradural spinal root myelin. Samples were fixed with glutaraldehyde, exposed to osmium tetroxide, embedded, thin sectioned, and finally stained with uranyl‐acetate and lead citrate. Reconstructions up to 4.2 nm resolution showed a non‐uniform distribution of stain in the planes of individual cytoplasmfc appositions (major dense lines). In each reconstructed major dense line the stain was distributed in striated, well‐defined structures. Those structures appear to be nearly parallel between neighboring major dense lines. The distribution of stain in the Schmidt‐Lanterman cleft did not resemble the distribution of stain in the major dense line; however, weak striations were present. Evidence that the striated structures are not an artifact due to image calculation is discussed.
Substratum‐induced modulation of acetylcholinesterase activity in cultured dorsal root ganglion neurons
Authors: Gupta J. J. , Bigbee J. W. 
Published: May 2005

AbstractAcetylcholinesterase (AChE) has been shown to be transiently expressed in the developing nervous system during periods of neuronal migration and axonal outgrowth. We are investigating the possible interaction of substratum with AChE activity in dorsal root ganglion neurons (DRGN) cultured on substrata with varying degrees of permissiveness for neurite outgrowth: (1) extracellular matrix substrata: reconstituted basal lamina Matrigel™ (MGEL), laminin (LAM) and type I collagen (COL), and (2) organotypic substrata: unfixed, frozen sections of sciatic nerve (SN) and spinal cord (SC). In group 1, histochemical staining for AChE in DRGN was lowest on MGEL where outgrowth was most vigorous, intermediate on LAM, and highest on COL where neurite outgrowth was reduced by 55 % compared to Matrigel™ and highly fasciculated. A similar trend was seen when the cultures were assayed biochemically, 2.84 ± 0.14 nmoles ACh hydrolyzed/ganglion/ hr (MGEL), 4.42 ± 0.19 (LAM), 5.79 ± 0.37 (COL). In group 2, SN supported an expansive outgrowth with lower AChE activity than in DRGN grown on SC where outgrowth was minimal. These studies show that the levels of AChE activity can be modulated by substratum, perhaps in proportion to the permissiveness of the substratum to neuritic outgrowth. These results are discussed in relation to possible non‐cholinergic roles of AChE.
Nerve growth factor and choline acetyltransferase activity levels in the rat brain following experimental impairment of cerebral glucose and energy metabolism
Authors: Hellweg R. , Nitsch R. , Hock C. , Jaksch M. , Hoyer S. 
Published: May 2005

AbstractIntracerebroventricular (ICV) injection of streptozotocin (STZ) has been reported to impair cerebral glucose utilization and energy metabolism (Nitsch and Hoyer: Neurosci Lett, 128:199–202, 1991) and also to prejudice passive avoidance learning in adult rats (Mayer et al.: Brain Res 532:95–100, 1990). It is well established that the forebrain cholinergie whose integrity is essential for learning and memory functions, depends on the target‐derived retrograde messenger nerve growth factor (NGF). Therefore, we measured NGF and choline acetyltransferasc (ChAT) activity levels in the forebrain cholinergic system in adult rats that had received a single injection of either STZ or artificial cerebrospinal fluid into the left ventricle 1 or 3 weeks prior to sacrifice. One week after ICV STZ treatment, NGF content was significantly decreased (−32%) in the septal region, where NGF‐responsive cell bodies are located and NGF exerts its neurotrophic action after retrograde transport from NGF‐producing targets. In contrast, NGF levels in the cortex and hippocampus, which are target regions for the basal forebrain cholinergic neurons, and in the brainstem and cerebellum were increased ( + 12% to +47%) within 3 weeks after ICV STZ treatment. The alterations in NGF levels were not related to changes in ChAT activity that decreased in the hippocampus by only 15%. This might be due to maskling effects exerted by compensatory NGF‐mediated stimulation of ChAT activity in remaining functional neurons. It is suggested that impaired behavior which fnas been observed after STZ‐induced impairment of cerebral glucose and energy metabolism may be at least partially related to a diminished capacity of cental NGF‐responsive neurons to bind and/or transport NGF. Damage of the cerebral glucose and energy metabolism by ICV treatment with STZ may therefore provide an appropriate animal model for the study of the early metabolic events in brain abnormalities associated with cognitive impairments.
cDNA cloning and chromosome assignment of the gene for human brain 14–3–3 protein η chain
Authors: Ichimura‐Ohshima Y. , Morii K. , Ichimura T. , Araki K. , Takahashi Y. , Isobe T. , Minoshima S. , Fukuyama R. , Shimizu N. , Kuwano R. 
Published: May 2005

AbstractWe present the nucleotide sequence of a cDNA clone of mRNA encoding human 14‐3‐3 protein, a protein kinase‐dependent activator of tyrosine and tryp‐tophan hydroxylases and an endogenous inhibitor of protein kinase C. The 1,730‐nucleotide sequence of the cloned cDNA contains 191 bp of a 5′‐noncoding region, the complete 738 bp of coding region, and 801 bp of a 3′‐noncoding region containing three canonical polyadenylation signals. The 14‐3‐3 protein η chain cDNA encoded a polypeptide of 246 amino acids with a predicted molecular weight 28,196. The predicted amino acid sequence of human 14‐3‐3 protein η was highly homologous to that of previously reported bovine and rat 14‐3‐3 proteins with only two amino acid differences. The sequence carries structural features as putative regions responsible for activation of tyrosine and tryptophan hydroxylases and for inhibition of Ca2+/phospholipid‐dependent protein kinase C. Northern blot analysis demonstrated widespread expression of the 14‐3‐3 protein η chain in cultured cell lines derived from various human tumors. These findings suggest the conservative functions of the 14‐3‐3 protein among species. Spot blot hybridization analysis with flow‐sorted chromosomes it showed that the human 14‐3‐3 protein η chain gene is assigned to chromosome 22.
An immortalized cell line expresses properties of activated microglial cells
Authors: Bocchini V. , Mazzolla R. , Barluzzi R. , Blasi E. , Sick P. , Kettenmann H. 
Published: May 2005

AbstractMurine cultured microglial cells were immortalized after infection with a v‐raf/v‐myc recombinant retro‐virus. This immortalized cell line (BV‐2) shares properties with body macrophages with respect to the antigen profile, their phagocytic capacity and antimicrobial activity. BV‐2 cells are not constitu‐tively able to kill tumor cells in vitro, but acquire antitumor activity following an increase in [Ca++]i. BV‐2 cells, like microglial cells, are however, distinct from peripheral macrophages by their expression of inwardly rectifying K+ cannels in concert with a Jack in outwardly rectifying K+ channels and the formation of spineous processes. The BV‐2 cell line thus represents a suitable model for in vitro studies of activated microglial cells.
Isolation and characterization of neonatal schwann cells from cryopreserved rat sciatic nerves
Authors: Mason P. W. , Attema B. L. , DeVries G. H. 
Published: May 2005

AbstractMuch of our knowledge about the development and maintenance of the peripheral nervous system has been learned through studying the interaction of neurons, or their isolated membranes, with Schwann cells (SC), in tissue culture. Numerous approaches have been employed to obtain an adequate quantity of SC, but all have been limited by either the uncertainty of obtaining a sufficient amount of starting material, the time and expertise required to isolate the SC, or by the limited number of SC that can be generated. We have developed a procedure to isolate SC from cryopreserved sciatic nerves. This procedure allows for sciatic nerves to be pooled until adequate numbers of nerves are obtained, yet still produces cells that retain the functional abilities of SC isolated from fresh nerves. Sciatic nerves were isolated from 2 day old rat pups, placed in either DME media and used fresh or placed in a freezing solution containing DME media (25%), DMSO (25%), fetal calf serum (50%), frozen at −70°C and stored in liquid nitrogen. The frozen nerves were rapidly thawed to 37°C and single cells were prepared from both fresh and frozen nerves using enzymatic and mechanical disruption as previously described (Brockes et al., Brain Res 165:105–118, 1979). Comparable cell yields were obtained for SC isolated from both frozen and fresh nerves. Immunohistochemical staining of both fresh and frozen SC produced similar staining patterns with antibodies to GFAP, laminin, CNPase, S100, MBP, and Po protein. Addition of axolemmal enriched membrane fractions to both the frozen and fresh SC gave a similar dose response curve of 3H‐thymidine incorporation, with SC from frozen sciatic nerves responding even better than fresh sciatic nerves at higher doses (50 μg and 100 μg of protein/ml). As demonstrated by the cell yield, immunohistochemical staining and responses to axolemmal mitogens, this procedure produces SC from frozen sciatic nerves with similar characteristics to those isolated from fresh nerves. This procedure will allow the production and utilization of a large number of SC, which will be critical in further studies on the development and maintenance of the peripheral nervous system.
Isolation of cDNA clones encoding rat glial fibrillary acidic protein: Expression in astrocytes and in Schwann cells
Authors: Feinstein D. L. , Weinmaster G. A. , Milner R. J. 
Published: May 2005

AbstractGlial fibrillary acidic protein (GFAP) expressed by astrocytes in the central nervous system (CNS) has been extensively characterized but the molecular identity of related molecules in the peripheral nervous system (PNS) remains unclear. To examine possible structural differences between CNS and PNS GFAP, we have isolated cDNA clones for rat GFAP from both cultured astrocyte and Schwann cell libraries. Nucleotide sequence analysis indicated that the PNS and CNS GFAP clones contained identical coding regions, with a predicted protein product of 430 amino acids. However, the 5′‐untranslated region of clone rGFA15, isolated from the Schwann cell library, was longer than that predicted for brain‐derived GFAP mRNA. Primer extension analysis of RNA isolated from the RT4‐D6 Schwann cell line indicated that the start site for PNS GFAP mRNA lies 169 bases upstream from that used in the CNS. In addition, tryptic peptide mapping of GFAP prepared from cultured astrocytes and Schwann cells revealed one major peptide fragment present in CNS GFAP but absent from PNS GFAP. These results suggest structural differences between GFAP in these two cell types, at both the nucleic acid and protein level, and are consistent with previous observations of immunochemical differences existing between CNS and PNS GFAP. © 1992 Wiiey‐Liss, Inc. © 1992 Wiley‐Liss, Inc.
Expression cloning of neurotrophic factors using Xenopus oocytes
Authors: Lam A. , Kloss J. , Fuller F. , Cordell B. , Ponte P. A. 
Published: May 2005

AbstractWe have explored the potential for cloning novel neu‐rotrophic factor cDNAs via assay of neurotrophic activities following expression in Xenopus oocytes. In this report, we describe the successful application of the method to tract rat ciliary neurotrophic factor (CNTF) activity from mRNA purified from cultured cells and from mRNA synthesized by in vitro transcription of a cDNA library. Rat C6 glioma cells, which had been previously shown to have CNTF‐like activity (Westermann et al., 1988), were used as source material. We tested protein extracts of C6 cells using an in vitro assay of primary neurons from the chick ciliary ganglion (CCG assay) and detected a CNTF‐like activity. RNA isolated from C6 cells was shown to direct the synthesis of the activity following microinjection into Xenopus oocytes and one‐step fractionation of Xenopus extract. C6 mRNA was size‐fractionated, and fractions encoding CNTF‐like activity were cloned into a lambda phage vector at a site distal to a T7 promoter. Synthetic RNA transcribed from total library DNA was injected into Xenopus oocytes, and a CNTF‐like activity in the oocyte extract was detected by the CCG assay. Further fractionation of library clones narrowed the presence of the clone encoding the CNTF‐like activity to a pool containing 20,000 members. The presence of a fulllength CNTF cDNA clone in this pool and partial clones in other pools was confirmed by Polymerase Chain Reaction (PCR) using oligonucleotides from the rabbit CNTF cDNA (Lin et al., 1989) as primers. A pure clone was isolated, shown by sequence analysis to have near identity to a published rat CNTF sequence (Stockli et al., 1989) and shown by the CCG assay to produce potent CNTF activity. The method described here does not require any knowledge of protein or DNA sequences; therefore, it is useful in cloning unknown neurotrophic activities. © 1992 Wiley‐Liss, Inc.
Specific interaction of glutamate with membranes from cultured retinal pigment epithelium
Authors: López‐Colomé A. M. , Salceda R. , Fragoso G. 
Published: May 2005

AbstractExcitatory amino acids (EAA) have been shown to induce phagocytosis in retinal pigment epithelial (RPE) cells. In order to explore if this action is receptor‐mediated, we have identified and characterized receptors for L‐glutamate through the binding of [3H]L‐glutamate to membranes from chick RPE cells in primary culture. Specific binding was found saturable, with KB = 333nM and Bmax = 3.2 pmol/mg protein in frozen/thawed membranes. Na+‐independent binding was present in cultures of 16 and 25 days in vitro, and was not affected by temperature. Pharmacological profile of analogues of EAA at different receptor types suggests the presence of a metabotropic type receptor (L‐glutamate > S‐2‐amino‐3‐phosphonopropionate > 2‐amino‐4‐phosphonobutyrate = trans‐(1S,3R)‐1‐aminocyclopentane‐1,3‐dicarboxylate > quisqualate). Excitatory amino acid analogues acting at the NMDA‐receptor also displaced bound L‐glutamate, and a noticeable stimulation of specific binding of this ligand by glycine was shown; this effect was mimicked by D‐serine and 1‐hydroxy‐3‐aminopyrrolidone‐2 (HA‐966) but not by 7‐chlorokynurenate, and was not inhibited by strychnine. Since taurine and GABA also increased specific binding, it is likely that modulation of EAA receptors in RPE differs from that in neurons. © 1993 Wiley‐Liss, Inc.
Manganese induces spreading and process outgrowth in rat pheochromocytoma (PC12) cells
Authors: Lin W. H. , Higgins D. , Pacheco M. , Aletta J. , Perini S. , Marcucci K. A. , Roth J. A. 
Published: May 2005

AbstractMn2+ has been shown to promote cell–substrate adhesion and cell spreading in many cell culture systems. In this study, we present data demonstrating that Mn2+ not only promotes spreading, but also induces process outgrowth in rat pheochromocytoma (PC12) cells. In the presence of 1.0 mM MnCl2, cell spreading was apparent by 6 hr, and nearly 50% of the exposed cells extended neurite‐like processes. These morphological effects of Mn2+ were both time‐ and dose‐dependent. In the presence of cycloheximide, a protein synthesis inhibitor, both Mn2+‐induced spreading and neurite outgrowth were prevented, indicating that de novo protein synthesis is required for the effects of Mn2+ to take place. Of the other divalent cations tested, Mg2+, Cd2+, Cu2+, Ni2+, and Zn2+ were ineffective, and only Co2+ partially mimicked the effects of Mn2+. Although Mn2+‐induced cell adhesion and spreading have been extensively studied, this is the first report that this divalent cation can cause neurite outgrowth. The neurite outgrowth‐promoting effects of Mn2+ were distinct from those of nerve growth factor in that the response to Mn2+ was considerably more rapid, but apparently lacked the ability to sustain continuous outgrowth and networking of neurites. Mn2+ also induced the levels of GAP‐43 and peripherin, two proteins associated with neuronal differentiation of PC‐12 cells. In cells grown in serum‐free defined medium, Mn2+ was capable of promoting neurite outgrowth when the cells were plated on surfaces pretreated with normal growth medium, vitronectin, or fibronectin, while it failed to cause these morphological changes in cells plated on untreated or poly‐D‐lysine‐coated substrata. Similarly, Mn2+ also promoted neurite outgrowth from rat sympathetic neurons attached to laminin‐treated substrate, but had no effect on neurons maintained on substrate with polylysine only. The pentapeptide Gly‐Arg‐Gly‐Asp‐Ser nearly completely prevented the morphological effects of Mn2+ on PC12 cells. These findings are consistent with a hypothesis that Mn2+‐mediated alteration of an RGD‐dependent extracellular matrix‐integrin interaction is responsible for the neuritogenic effects. © 1993 Wiley‐Liss, Inc.
Blockage of nicotinic acetylcholine receptors by 5‐hydroxytryptamine
Authors: Grassi F. , Polenzani L. , Mileo A. M. , Caratsch C. G. , Eusebi F. , Miledi R. 
Published: May 2005

AbstractThe action of 5‐hydroxytryptamine (5HT) on nicotinic acetylcholine receptor (nAChR) channels was investigated in mouse myotubes, human cloned TE671/RD cells, and Xenopus laevis oocytes. The decay of the ACh‐activated whole‐cell currents was reversibly accelerated in the presence of 5HT (10−5 to 10−3 M), in a dose‐dependent manner. 5HT also reduced the size and accelerated the decay of currents elicited by ACh in Xenopus oocytes injected with mRNA extracted from C2 myotubes or Torpedo electroplaques, or oocytes injected with cloned mouse muscle AChR subunit mRNAs. The effect of 5HT was promptly reversed after washout, or by depolarizing the oocyte beyond −10 mV. In patch‐clamp recordings from myotubes, bath‐application of 5HT did not exert an indirect influence on the ACh‐activated channels within the patch membrane. In contrast, when the patch membrane was exposed to 5HT (10−6 M), ACh unit responses appeared as bursts of short pulses. It is concluded that the regulation of ACh responses by 5HT results from a fast noncompetitive blocking action of nAChR‐channels. These results show that ligand‐gated channels, activated by their specific neurotransmitter, may be regulated by a different neurotransmitter through a direct action on the receptor molecule. © 1993 Wiley‐Liss, Inc.