bluuebunny/crossref_metadata_2025_split huggingface dataset:
Organization of oligodendroglial membrane sheets. I: Association of myelin basic protein and 2′,3′‐cyclic nucleotide 3′‐phosphohydrolase with cytoskeleton
Authors: Dyer C. A. , Benjamins J. A. 
Published: January 2005

AbstractMembrane sheets elaborated by cultured murine oligodendroglia provide a unique system for examining associations between myelin proteins and cytoskeletal elements. Interactions can be observed and manipulated more readily than in the multilamellar myelin membrane in vivo. Immunocytochemical staining of 2′,3′‐cyclic nucleotide 3′‐phosphohydrolase (CNPase) shows that it is distributed diffusely in some regions of membrane sheets, but colocalized with tubulin in lacy networks and major veins in other regions. Staining with phalloidin also reveals two distributions of F‐actin: (1) small aggregates within the diffuse CNPase regions and (2) filaments colocalized with tubulin and CNPase in the lacy networks and veins. Application of colchicine at 10 μg/ml for 4 hr disrupts microtubular structures in the lacy network, while those in major veins remain intact. This suggests that microtubules in the lacy network are treadmilling more rapidly than those in the major veins. The distribution of CNPase and F‐actin is not altered under these conditions. In contrast, cytochalasin B disrupts F‐actin, microtubules, and CNPase in the lacy networks, indicating that cross‐linking between these three proteins is disrupted. Both colchicine and cytochalasin B cause fusion of myelin basic protein (MBP) domains in membrane sheets. This appears to be a consequence of disruption of microtubules in the lacy networks, which normally outline the MBP domains. In summary, these results provide evidence for (1) direct association of CNPase with F‐actin and tubulin in cytoskeletal structures and (2) organization of MBP into domains via association with microtubules in the lacy networks.
Glycerophosphorylcholine phosphocholine phosphodiesterase activity of rat brain myelin
Authors: Kanfer J. N. , McCartney D. G. 
Published: January 2005

AbstractMyelin isolated from rat brain possessed the ability to release phosphorylcholine from glycerophosphoryl‐choline, and this activity was enriched 3.2‐fold over that of the original homogenate. This glycerophosphorylcholine phosphocholine phosphodiesterase activity had a pH optimum at 9.5, had a Km of 0.2 mM, and a Vmax of 150 nmoles/mg protein/hr. The enzyme had a specific requirement for Zn2+ with an optimum concentration at 0.25 mM. Maximum enzyme activity was at 50°C and an Arrhenius plot showed a breakpoint at 40°. p‐Nitrophenylphosphorylcholine was also hydrolyzed by purified myelin and was a competitive inhibitor of glycerophosphorylcholine phosphocholine phosphodiesterase activity with a Ki of 0.075 mM. Glycerolphosphorylethanolamine was hydrolyzed only 5% compared with GPC, but it was not an inhibitor.
Resistance to extrapyramidal effects of opiates in rats chronically treated with SCH 23390
Authors: De Montis G. M. , Devoto P. , Meloni D. , Porcella A. , Saba P. , Tagliamonte A. 
Published: January 2005

AbstractRats made tolerant to morphine show neither a change in brain opiate receptor number nor altered sensitivity to the inhibitory effect of opiates on striatal adenylate cyclase (AC) activity. Interestingly, SCH 23390, a selective blocker of D1 dopamine (DA) receptors which, given chronically to rats, induces a 32% increase in D1 receptor number and increases the Vmax of D1‐stimulated striatal AC, resulted in marked resistance to acute morphine effects. In particular, rats chronically treated with SCH 23390 failed to show muscular rigidity and increased striatal dihydroxyphenylacetic acid (DOPAC) concentration after morphine. Moreover, basal striatal AC activity in these animals had a significantly reduced sensitivity to opiate inhibition.On the other hand, decreased AC sensitivity to acetylcholine (ACh) inhibition observed in the striatum of rats chronically treated with DFP, an irreversible blocker of acetylcholinesterase, appeared to be secondary to the downregulation of muscarinic receptors and thus did not modify the opiate inhibitory capacity. It was concluded that although a potentiation of striatal AC impairs opiate action, such mechanism is not involved in morphine tolerance.
Cyclic adenosine monophosphate as a second messenger in horizontal cell uncoupling in the teleost retina
Authors: Laufer M. , Salas R. , Medina R. , Drujan B. 
Published: January 2005

AbstractThe reduction in the receptive field of horizontal cells of the teleost Eugerres plumieri observed upon dopamine (DA) superfusion is thought to be due to cell uncoupling. The possible mechanisms by which activation of DA receptors modify the electric coupling between horizontal cells were studied in the present work. It was found that the effect of DA in different preparations is mediated by a modification of intracellular concentration of cAMP and H+. The effects of intracellular injection of cAMP and H+ were studied in retinal horizontal cells of the teleost E. plumieri. A triple microelectrode was used to inject the ion iontophoretically, to pass current pulses, and to record voltages from the same cell, while a fourth microelectrode was used to record voltages from a neighboring cell in the same retinal layer. Responses evoked by light spots and annuli were evaluated simultaneously.Coupling ratios between neighboring horizontal cells ranged from 0.22 to 0.45. The intercellular resistance (Rc), 0.5–3.5 × 106 ohms, and that of the remaining cell membrane resistance (Rm), 2.5–18 × 106 ohms, were calculated by means of a passive electrical model that has a hexagonal array.The microinjection of H+ with injection current from +5 to +30 nA for 40 to 100 sec led to temporary and reversible light response reduction. The coupling ratio between two impaled cells was reduced by about 30%, and intercellular resistance (Rc) increment was 320% while cell membrane resistance (Rm) did not change consistently.There was also a temporary and reversible Rm reduction (70–85%) and an Rc increment of 170–330% when cyclic adenosine monophosphate was iontophoretically injected with current from −30 to −40 nA for 50 to 170 sec. The coupling ratio between two impaled cells was reduced by about 40%, and light responses recorded from the injected cell showed a reduction in amplitude with the same time course as that of the resistive changes. The injection of Lucifer yellow into a horizontal cell under normal conditions always results in pronounced fluorescence for more distant cells; however, under constant injection of H+ or cAMP only the injected cell is fluorescent, which provides direct evidence of the reduction in the effectiveness of coupling between horizontal cells. The observed effects of intracellular H+ or cAMP injection correspond to the resistive changes in Rc and coupling ratio that occur in the horizontal cell network upon superfusion with a dopamine (DA) solution.
Survival of purified embryonic chick retinal ganglion cells in the presence of neurotrophic factors
Authors: Lehwalder D. , Jeffrey P. L. , Unsicker K. 
Published: January 2005

AbstractIn a search for neurotrophic factors (NTFs) regulating retinal ganglion cell (RGC) death in the chick embryo we have used purified and cultured RGCs. Purification of RGCs from embryonic day 10 was achieved by employing the “panning” method (Silverstein and Chun: Soc Neurosci Abstr 13:1054, 1987). The obtained neuron population consisted of 97% RGCs as demonstrated by retrograde labeling with a fluorescence dye. RGCs were cultured at low density in a chemically defined medium and shortterm survival (24 hr) was determined. In the absence of NTFs, less than 3% of the RGCs survived. In the presence of various crude or purified NTFs (eye, brain, and tectum extracts; glial‐conditioned medium; ciliary nerotrophic factor [CNTF]; nerve growth factor [NGF]) 31% to 52% of the RGCs were maintained. The effects of NGF and CNTF were not additive. Neither acidic nor basic fibroblast growth factor was able to maintain RGCs in culture. Our results, obtained with a culture system which allowed the analysis of direct trophic actions, suggest that NGF and CNTF may be NTFs for overlapping subpopulations of chick RGCs.
Aminoglycoside antibiotics impair calcium entry but not viability and motility in isolated cochlear outer hair cells
Authors: Dulon D. , Zajic G. , Aran J.‐M. , Schacht J. 
Published: January 2005

AbstractCochlear outer hair cells have been well established as primary targets of the ototoxic actions of aminoglycoside antibiotics. These cells, isolated from the guinea pig cochlea and maintained in short‐term culture, were used as a model for evaluating the acute effects of gentamicin on cell viability, depolarization‐induced transmembrane calcium flux, and depolarization‐induced motile responses.On the basis of morphology and fluorochromasia, the presence of extracellular gentamicin as high as 5 mM did not affect the viability of the cells for up to 6 hr, the longest time tested. Viable cells showed binding of fluorescently tagged gentamicin to their base but excluded the drug from their cytoplasm. In response to [K+]‐depolarization, intracellular calcium levels (monitored with the fluorescent calcium‐sensitive dye fluo‐3) increased from a resting value of 218 ± 102 nM to 2,018 ± 1,077 nM concomitant with a cell shortening of 0.7% ± 1.3%. The depolarization‐induced calcium increase was apparently caused by calcium entry into the cell as it was inhibited by the calcium‐channel blocker methoxyverapamil and prevented in the absence of extracellular calcium. Both gentamicin and neomycin blocked the [K+]‐induced calcium increase at an IC50 of 50 μM. Despite the inhibition of calcium entry the ability of the outer hair cells to shorten under [K+]‐depolarization was not impaired; in fact, cell shortening was even more pronounced in the absence of calcium influx (2.6% ± 1.4%). This argues effectively against the existence of a calcium‐dependent actomyosin‐mediated component in [K+]‐induced shape changes.The results suggest the existence of voltage‐gated calcium channels in outer hair cells and that calcium influx through these channels is impaired by the aminoglycoside antibiotics neomycin and gentamicin. This action may be part of the acute ototoxic mechanism of these molecules. Furthermore, the results not only confirm the calcium independence of the depolarization‐induced motility but also suggest that calcium influx into outer hair cells opposes cell shortening.
Expression of synaptophysin and neuron‐specific enolase during neuronal differentiation in vitro: Effects of dimethyl sulfoxide
Authors: Schilling K. , Barco E. Blanco , Rhinehart D. , Pilgrim Ch. 
Published: January 2005

AbstractNeural development in dissociated cell cultures of fetal rat brain can be expected to depend on synaptic interactions between cultured neurons. Therefore, an attempt was made to obtain a quantitative measure of the time course of synaptogenesis in such a culture system by assessing the level of the secretory vesicleassociated protein synaptophysin (p38). The developmental schedule of p38 was compared to that of neuron‐specific enolase (NSE), an established marker of neuronal differentiation.Cultures were raised from dissociated 14 day‐old fetal rat diencephalon. In cultures grown for 1–2 days in vitro (DIV), p38‐immunoreactivity was preferentially located in neuronal perikarya. After 10–16 DLV, neurons in culture had formed a dense neuritic network, and almost all of the p38‐immunoreactivity occurred in the form of fine punctate deposits associated with neuronal processes that often outlined neuronal cell bodies in a basket‐like fashion. Electron‐microscopic immunocytochemistry proved the punctate deposits to be presynaptic elements, mostly in the form of axonal varicosities. Quantitative immunoblotting showed that levels of p38 increased from the start of cultivation to DIV 4, stayed fairly constant from DIV 4 to DIV 8, and rose again steeply to peak at DIV 12. In contrast, levels of NSE rose continuously up to DIV 12. After DIV 12, levels of both p38 and NSE fell again.Treatment of cultures with dimethyl sulfoxide (DMSO), an agent known to induce differentiation in various normal and malignant cell types, resulted in a significant increase of p38 levels and in a decrease of NSE levels. The amount of p38 continued to increase beyond DIV 12, whereas NSE diminished after having reached a maximum at DIV 12.Assessment of p38 levels as a measure to quantify synaptogenesis in culture has been validated through combined morphological observations and quantitative immunoblotting. The results show that expression of NSE and p38 during neuronal development follows different time schedules and that the two neuron‐specific proteins can be manipulated independently from each other. DMSO treatment appears to selectively promote synaptogenesis and/or maturation of specific synaptic properties.
Denervation and hyperinnervation in the nervous system of diabetic animals. I. The autonomic neuronal dystrophy of the gut
Authors: Di Giulio A. M. , Tenconi B. , La Croix R. , Mantegazza P. , Cattabeni F. , Gorio A. 
Published: January 2005

AbstractPeripheral neuropathy is a correlate of experimental diabetes induced in rats by means of a single injection of alloxan. The autonomic and enteric innervation of the gut are profoundly affected in the small intestine of such animals. A complex process of denervation and hyperinnervation of the gut wall of diabetic animals is observed. It was previously reported that the cholinergic parasympathetic innervation of the intestine is markedly reduced. We have found that noradrenergic sympathetic axons hyperinnervate the duodenum of diabetic rats, whereas noradrenaline levels are significantly reduced in the jejunum. The putative enteric neurotransmitter dopamine is also present in higher levels in the duodenum. The intrinsic peptidergic neurons of the gut are deeply affected as well in diabetic rats. Substance P and met‐enkephalin content are remarkably reduced throughout the small intestine, whereas vasoactive intestinal polypeptide levels (VIP) are significantly increased in the duodenum. Indeed, immunocytochemical staining of the ileum did reveal hypertrophy of VIP‐positive axons in diabetic rats. The intrinsic serotoninergic innervation of the gut is apparently unaffected. Our results indicate that the changes of gut innervation observed in experimental diabetes are consistent with increased content and also likely with hyperinnervation by the neuronal systems involved in smooth muscle relaxation and decreased content and with denervation by those systems with smooth muscle contraction properties. Such a perturbed gut innervation may be responsible of the gastrointestinal dysfunctions that are among the most common complications of diabetes.
Phosphatidate phosphohydrolase in purified rat brain myelin
Authors: Vaswani K. K. , Ledeen R. W. 
Published: January 2005

AbstractHighly purified myelin from rat brain stem has been shown to contain phosphatidate phosphohydrolase, an enzyme which converts phosphatidate to diacylglycerol. The high levels relative to cytosol and microsomes (17% and 22%, respectively) tended to preclude contamination by these fractions as the source of activity. Additional evidence came from study of repeated purification, mixing experiments, and washing of the myelin with salt and detergent. We conclude that this enzyme, in addition to being widely distributed in other subcellular fractions, is intrinsic to the myelin membrane. Through its activity it generates a key substrate for the cytidine (Kennedy) pathway which was previously shown to occur in this membrane.
Glucocorticoids modulate rat hypothalamic corticotrophin‐releasing factor release induced by interleukin‐1
Authors: Cambronero J. C. , Borrell José , Guaza C. 
Published: January 2005

AbstractContinuous in vitro perifusion of rat hypothalami with interleukin‐1 β (IL‐1) increased corticotropinreleasing factor (CRF) secretion in a dose‐dependent manner, in the range of 1 to 5 U/ml. The stimulatory action of IL‐1 was significantly attenuated by dexamethasone, both by addition to the perifusion medium and by in vivo dexamethasone pretreatment. The data suggest that an immunoneuroendocrine interaction circuit, subjected to glucocorticoid negative feedback, has an essential role in maintaining organism homeostasis.
Glial cells of the oligodendrocyte lineage express proton‐activated Na+ channels
Authors: Sontheimer H. , Perouansky M. , Hoppe D. , Lux H. D. , Grantyn R. , Kettenmann H. 
Published: January 2005

AbstractNeurons and oligodendrocytes, but not type I astrocytes and Schwann cells, generate large Na+ currents in response to a step increase of [H+]. Proton‐activated Na+ channels are the first cationic channels expressed in neuronal precursor cells from the mammalian brain. Glial precursor cells cultured from mouse brain are also capable of generating Na+ currents in response to step acidification (INa(H)). With further development along the oligodendrocyte lineage, this property is retained, whereas voltage‐activated Na+ and K+ currents disappear. Comparing INa(H) of oligodendrocytes with INa(H) of their precursor cells did not reveal a difference in current amplitude, suggesting a higher density of INa(H) channels on the (smaller) precursor cells. The properties of INa(H) in glial precursor cells and oligodendrocytes are similar to those of neurons, with respect to activation conditions, time course, and the effect of extracellular Ca2+ concentrations. The results are consistent with previous observations which showed that oligodendrocytes partially preserve their chemically activated, but completely lose their voltage‐activated, ion channels.
Binding properties of liposomes containing the myelin‐associated glycoprotein MAG to neural cell cultures
Authors: Sadoul R. , Fahrig T. , Bartsch U. , Schachner M. 
Published: January 2005

AbstractThe myelin‐associated glycoprotein MAG is a neural cell adhesion molecule which belongs to the immunoglobulin superfamily and the carbohydrate based L2/HNK‐1 family of adhesion molecules. In this study we further characterize the adhesive properties of MAG. MAG incorporated into liposomes bound to cultured peripheral and central nervous system neurons known to be myelinated in vivo. Expression of the neuronal MAG receptor(s) on spinal cord neurons increased with time in culture and correlated with the time of active myelination of these neurons in vivo. MAG bound only poorly if at all to cerebellar neurons which are not myelinated in vivo and not to cultured oligodendrocytes or Schwann cells. A low level of MAG binding to astrocytes or fibroblast‐like cells that was MAG antibody molecules L1 and N‐CAM, two other members of the immunoglobulin superfamily, were not found to be the neuronal receptors for MAG. RGD containing peptides did not inhibit binding of MAG‐liposomes to neurons. The soluble form of MAG which contains most, if not all, of the extracellular domain of the molecule and binds to collagen, did not interfere with the binding of MAG‐liposomes to neurons. Conversely, MAG‐liposomes did not bind to collagen, suggesting the extracellular domain of the molecule.
Dual‐label retrograde transport: CNS innervation of the mouse thymus distinct from other mediastinum viscera
Authors: Tollefson L. , Bulloch K. 
Published: January 2005

AbstractTwo different retrogradely transported tracers were identified and used to distinguish between the innervation of the thymus gland and the innervation of adjacent tissues residing within the mouse mediastinum. To determine the best procedure for peripheral dual‐labeled retrograde transport, several different tracers, vehicles, and injection protocols were developed and/or tested. In the periphery, it proved ineffective to use Rhodamine latex microspheres (Luma‐Fluor) as a retrograde transport agent. Horseradish peroxidase (HRP) suspended in the water‐soluble carrier KY Jelly and Fluro‐gold (FG) in an aqueous solution was the most effective combination of retrograde transport tracers. Equivalent doses of each of these tracers were determined, and injections of HRP or FG into the thymus and the esophagus produced distinct and separate patterns of labeled neurons in the spinal cord or brain stem. Throughout these CNS areas, some neurons contained both tracers suggesting that (1) these areas are innervated by neurons that give rise to collaterals to these tissues, (2) that the retrograde tracers are transported by nerves that innervate the lymphatics of the mediastinum, and/or (3) that some diffusion of the tracers occurs from the test organs and tissues to other organs and tissues of the mediastinum.
Expression of transfected genes by differentiated, postmitotic neurons and photoreceptors in primary cell cultures
Authors: Werner M. , Madreperla S. , Lieberman P. , Adler R. 
Published: January 2005

AbstractWe report here that differentiated, primary, postmitotic neurons and photoreceptors in cultures obtained from embryonic chicks can express foreign genes after transfection by the calcium phosphate method. A variety of viral promoters were tested by using either β‐galactosidase or chloramphenicol acetyltransferase as reporter genes. Histochemical and immunocytochemical analysis showed β‐galactosidase expression by both neurons and photoreceptors. As commonly observed with dividing cells, transfection efficiencies showed inter‐experimental variability, with efficiencies ranging from 2% to 20% using the same plasmid. On the other hand, intra‐experimental variability between replicate dishes was much smaller. Analysis using the highly sensitive enzymatic assay for chloramphenicol acetyl transferase (CAT) showed that all of the promoter/enhancers‐CAT constructs tested, with the exception of a construct containing the Maloney sarcoma virus promoter, led to the expression of detectable activity when transfected into cultured retinal cells. The calcium phosphate treatment used for cell transactions did not show detectable effects on overall cell survival, although it caused selective decreases in some metabolic activities of the cells. The studies demonstrate that it is possible to obtain expression of genes transfected into primary, postmitotic neuronal cells.
Purification of an endogenous 68 kD inhibitor of calcium‐activated neutral proteinase (CANP) from bovine brain: Immunoblot identification and characterization
Authors: Banik N. L. , Chakrabarti A. K. , Hogan E. L. 
Published: January 2005

AbstractA calcium‐activated neutral proteinase (CANP)‐specific endogenous inhibitor (calpastatin) was purified from bovine brain by successive column chromatography. The purified inhibitor exhibited a major band on sodium dodecylsulfate polyacrylamide gel electrophoresis with an approximate molecular weight of 68 kD. The polyclonal antisera raised to the inhibitor strongly reacted with the 68 kD protein band. Two lightly stained bands approximately 55–68 kD and 120–130 kD were also recognized by the inhibitor antiserum. The inhibitor specifically inhibited CANP activity and the half‐maximal inhibition was found with 75 ng of calpastatin per 1 μg of CANP in a final volume of 125 μl. Cathepsin B and papain were not inhibited by the inhibitor, while trypsin and chymotrypsin were inhibited to some extent. The inhibitor formed a complex with CANP and the inactive complex was dissociated into active fractions of enzyme and calpastatin in the presence of EGTA.