bluuebunny/crossref_metadata_2025_split huggingface dataset:
Calcium/Calmodulin‐dependent protein kinase activity in primary astrocyte cultures
Authors: Babcock‐Atkinson E. , Norenberg M.D. , Norenberg L.O.B. , Neary J.T. 
Published: January 2005

AbstractCalcium, calmodulin‐dependent protein kinase (Ca/CaM kinase) is an important component of calcium signalling mechanisms in the brain, but little is known about the properties of this protein phosphorylation system in astrocytes. Addition of calcium and calmodulin to supernatant or membrane fractions obtained from rat astrocytes in primary culture increased phosphate incorporation into an exogenously added substrate, casein, and into endogenous protein substrates; this increase was greater than that observed with either calcium alone or calmodulin alone. The calcium, calmodulin‐stimulated increase was inhibited by trifluoperazine, and this inhibition could be overcome by the addition of excess calmodulin. The major substrates for Ca/CaM kinase activity were proteins with molecular weights of 59 and 53 kDa, which were similar, but not identical, to the subunits of Ca/CaM kinase type II from brain. The specific activity of Ca/CaM kinase and the phosphorylation of 59 kDa were increased in astrocyte cultures treated and maintained in dibutyryl cyclic adenosine monophosphate (dBcAMP). These results indicate that astrocytes contain Ca/CaM kinase activity and suggest an interaction between the cAMP and calcium/calmodulin messenger systems in these cells.
Evidence for an early role for myelin‐associated glycoprotein in the process of myelination
Authors: Owens Geoffrey C. , Bunge Richard P. 
Published: January 2005

AbstractThe expression of myelin‐associated glycoprotein (MAG) in purified rat Schwann cells following coculture with dorsal root ganglion neurons was compared with the expression of galactocerebroside (GalC) and Po using immunocytochemistry. In defined serum‐free medium, lacking ascorbic acid, in which Schwann cells proliferate but neither ensheathe nor myelinate axons, axonal interaction up‐regulated the cell surface expression of MAG and GalC but not of Po. Excision of neuronal cell bodies resulted in a down‐regulation of both MAG and GalC from the Schwann cell surface. When cocultures were switched to complete medium (serum plus ascorbic acid) to promote myelination, Schwann cells committed to form myelin continued to express high levels of MAG and GalC on their surface, but nonmyelinating Schwann cells down‐regulated MAG and GalC. There was significant MAG immunoreactivity associated with the external aspect of the apparent nodal region of developing myelin sheath. Permeabilization prior to immunostaining revealed that all of the Schwann cell cytoplasmic processes of nascent internodes were significantly stained with anti‐MAG antibodies before the appearance of Po immunoreactivity. The amount of MAG on the surface of mature myelin segments was reduced compared with developing myelin segments, but there was a considerable amount of anti‐MAG staining in the paranodes and Schmidt‐Lanterman incisures. The time of expression and localization of MAG indicates that it may be a critical molecule in the process by which the Schwann cell engulfs an axon destined to be myelinated and establishes the extent of the future internode.
Gliosis in the spinal cords of rats with experimental allergic encephalomyelitis: Immunostaining of carbonic anhydrase and vimentin in reactive astrocytes
Authors: Cammer Wendy , Tansey Francine A. , Brosnan Celia F. 
Published: January 2005

AbstractSpinal cord sections from rats sensitized to develop experimental allergic encephalomyelitis (EAE) were immunostained with antibodies against glial fibrillary acidic protein (GFAP), carbonic anhydrase, and vimentin, to see whether the latter two antigens could be detected in GFAP‐positive reactive astrocytes. Sixteen days after sensitization (16 dpi) there was intense carbonic anhydrase immunostaining in GFAP‐positive cells in the spinal cords of EAE rats, particularly in the white matter. At 13 and 20 dpi carbonic anhydrase immunostaining in astrocytes was less intense, and in the spinal cord white matter of control animals carbonic anhydrase was not detected in the few GFAP‐positive cells. In the spinal cords of EAE rats vimentin immunostaining was observed in inflammatory cells and astrocytes. In the latter, GFAP and carbonic anhydrase were colocalized with vimentin. The data suggest that carbonic anhydrase expression in astrocytes is an acute response to injury and that vimentin can be detected in astrocytes, as well as inflammatory cells, as early as 16 dpi.
Developmental accumulation and heterogeneity of myelin basic protein transcripts in the chick visual system
Authors: Zopf Dieter , Sonntag Vera , Betz Heinrich , Gundelfinger Eckart D. 
Published: January 2005

AbstractBy selective cloning of gene products newly expressed in the developing chick optic tectum, we have isolated cDNA clones encoding myelin basic protein (MBP). Three different transcripts of the corresponding single copy gene of 2.7 kb, 2.0 kb, and 1.1 kb were observed on Northern blots. In the developing optic lobe, the most abundant RNA (2.7 kb) was detected first at the 14th day in ovo. Nuclease S1 protection experiments revealed further heterogeneity of MBP transcripts, which result from alternative splicing of segments corresponding to the fifth and sixth exon of the homologous murine MBP gene. From these data, at least three different forms of MBP are predicted to exist in the chicken central nervous system.
Dynamic morphological responses of mouse astrocytes in primary cultures following medium changes
Authors: Devon Richard M. , Juurlink Bernhard H.J. 
Published: January 2005

AbstractWithin 5 minutes of changing the medium of 3–4 week old mouse astrocyte cultures, dramatic morphological alterations were seen in the cultures at the phase microscope level. These alterations included the appearance of membrane ruffles, followed by the formation of phase light vacuolar regions. Ultrastructurally, these ruffles could be seen as phagocytic arms, and the vacuoles appeared as blisters of the superficial cell layer. These morphological responses were due to the introduction of new serum proteins. Colloidal gold‐labelled serum proteins were used to visualize the dynamics of the macromolecular uptake following this medium change. Some proteins were taken up by phagocytosis, whereas others were associated with coated vesicles and endosomal vesicles. The majority of these gold‐labelled proteins were concentrated in vacuoles (presumably lysosomes) and were localized in the superficial cellular sheets. Inner cellular sheets contained little colloidal gold‐labelled serum proteins, but displayed prominent pinocytotic profiles. Glucose consumption in these cultures remained constant at 1.6 μmoles/mg protein/hr. Glycogen content varied among individual cells, but it remained constant in the cultures at 60 nmoles bound glucose/mg protein throughout the 48 hour examination period. These results suggest that dense cultures of primary astrocytes may act similarly to the glia limitans.
Chloride enters glial cells and photoreceptors in response to light stimulation in the retina of the honey bee drone
Authors: Coles Jonathan A. , Orkand Richard K. , Yamate Carol L. 
Published: January 2005

AbstractDouble‐barrelled ion‐selective microelectrodes were used to measure free [Cl−] in photoreceptors, extracellular space, and glial cells in superfused slices of drone retina. Tests indicated that with normal superfusate the intracellular electrode signal was due essentially to Cl− and not to some other interfering anion. The results indicate that Cl− is more concentrated in both photoreceptors and glial cells than would be predicted for a passive electrochemical distribution. When the photoreceptors were stimulated by a standard train of 20 ms flashes, 1/s for 90 s, their intracellular free [Cl−] (Cli) rose by 8 ± 1 mM. At the end of stimulation Cli usually continued to rise for up to a further 2 min and then returned toward the baseline over about 10 min. During light stimulation Cli in the glia rose. The magnitude of the increase was 5.1 ± 0.4 mM, about half the increase in Ki. In some extracellular recording sites, light stimulation caused [Cl−] to increase and in others to decrease. The mean change was –0.7 mM, SD 6.5 mM. The Cl− that entered the photoreceptors and the glia was presumably made available by the shrinking of the extracellular space. When the cells were depolarized by increasing [K+] in the superfusate from 7.5 mM to 18 mM, Cli increased. The half‐time of the change in Cli was longer than the half‐time of the depolarization by 10–30 s in the glia and 50–250s in the photoreceptors. During superfusion with 0 Cl− Ringer's solution, the light‐induced rise in extracellular [K+] was greater by a factor of 1.4–2.7, and the clearance after the end of the stimulation was slower. The rate of increase in glial Ki during light stimulation fell; the rate of increase of glial Ki caused by superfusion with raised [K+] (in the absence of Cl−) fell more. We conclude that when extracellular [K+] is increased, entry of Cl− into the glia is necessary for part, but not all, of the net uptake of K+. During light stimulation, the observed movement of Cl− into glia contributes to homeostasis of extracellular [K+], and the cell swelling associated with movement of Cl− into both glia and photoreceptors contributes to homeostasis of extracellular [Na+].
Bradykinin induces inositol 1,4,5‐trisphosphate‐dependent hyperpolarization in K+ M‐current‐deficient hybrid NL308 cells: Comparison with NG108–15 neuroblastoma × glioma hybrid cells
Authors: Higashida H. , Okano Y. , Hoshi N. , Yada Y. , Yokoyama S. , Asaga T. , Fu T. , Nozawa Y. 
Published: January 2005

AbstractExternal application of bradykinin (BK) to mouse neuroblastoma × mouse fibroblast hybrid NL308 cells and mouse neuroblastoma × rat glioma hybrid NG108–15 cells produced a transient outward (hyperpolarizing) current. In NG108–15 cells, BK also induced an inward (depolarizing) current associated with a decrease in input membrane conductance, which results from the inhibition of a voltage‐sensitive potassium current, the M‐current. However, in NL308 cells, either no depolarization was elicited by BK or, even if the BK‐induced depolarization was evoked, it was associated with an increased conductance. To explain the above difference, the intracellular second messenger system of NL308 cells was examined in detail. BK induced the rapid accumulation (three‐ to fivefold higher than the control level) of inositol 1,4,5‐trisphosphate (InsP3) in NL308 cells. The cytosolic Ca2+ concentration was also elevated to 540 nM from 180 nM at a basal level. This seems to be enough to activate a voltage‐independent and Ca2+‐sensitive K+ current, resulting in the hyperpolarization. Intracellular injection of InsP3 replicated the hyperpolarization. NL308 cells possess protein kinase C (C‐kinase), with specific activities of C‐kinase in cytosolic and membrane fractions being 233 and 24 pmol/min/mg protein, respectively. The activity associated with particulates became higher after phorbol dibutyrate (PDBu) treatment. But NL308 cells did not show the characteristic inward relaxation by step hyperpolarizations and the outward rectification in the current‐voltage relationship, indicating that the M current is deficient in NL308 cells. Therefore, application of PDBu failed to mimic the inward current. The results suggest the role of InsP3 and C‐kinase in controlling two K+ currents.
Ultrastructural changes in glial cells during regeneration of cockroach peripheral nerve
Authors: Blanco Rosa E. 
Published: January 2005

AbstractAfter peripheral nerve 5 in the cockroach Periplaneta americana was cut, changes occurring in the glial cells in the proximal stump were studied immediately after damage and during the process of nerve regeneration. During the first week haemocytes accumulated outside the nerve and morphologically similar granule‐containing cells appeared inside the nerve. These cells were involved in phagocytic activity. Between the second and the fourth week, signs of regeneration were distinguishable; many small axonal sprouts were formed which were surrounded by glial processes, and the nerve stump increased in length. During this period the glial cells produced large amounts of extracellular material in which the bundles of axons and glia were embedded. The structural differences between glial and perineurial cells were lost during these stages of regeneration and there was no restriction to the penetration of the extracellular tracer lanthanum. After 8 weeks, reinnervation of the muscles had taken place, perineurial and glial cells were again distinguishable, and the perineurial cells were able to exclude lanthanum.
Fibroblast growth factor inhibits epidermal growth factor‐induced responses in rat astrocytes
Authors: Huff Kenneth R. , Schreier Wayne 
Published: January 2005

AbstractThe signals which regulate the proliferation of astrocytes have relevance to normal developmental processes, transformational loss of growth control, and reactive gliosis present in many brain disease states. We have studied, in primary cultures of rat astrocytes, a sequential interaction of two growth factors, epidermal growth factor (EGF) and fibroblast growth factor (FGF), which may be relevant to the brain in these conditions. EGF is a strong mitogen and stimulator of 2 deoxyglucose (2 DG) transport with no effect on plating of cells, and FGF is a lesser mitogen and 2 DG uptake stimulator. However, when FGF is given to the cells as a pretreatment, FGF strongly inhibits the ability of EGF to stimulate both DNA synthesis and 2 DG uptake. The inhibition of EGF stimulation by FGF is across the EGF dose‐response curve, present at high and low culture densities, and stable for at least 3 days. Specificity is indicated by lack of inhibition by PDGF pretreatment and much less inhibition of fetal calf serum‐induced stimulations than EGF‐induced stimulation. Cell counts confirmed that the FGF pretreatment also inhibits EGF stimulation of cell division. Because of FGF brain derivation and angiogenic and neurotropic functions, it may serve as a regulator of EGF‐astrocyte interactions in processes such as development, gliomatous transformation, and neural regeneration.
Orthogonal arrays of particles in astroglial cells: Quantitative analysis of their density, size, and correlation with intramembranous particles
Authors: Neuhaus Jochen 
Published: January 2005

AbstractAstroglial cells were investigated by means of freeze‐fracture in normal rat and mouse brain, cell culture and human gliomas. Membranes of these cells were quantitatively analyzed for their intramembranous particles (IMPs) and orthogonal arrays of particles (OAPs). Measurement of the size of OAPs and IMPs has permitted the search for a correlation between the 7‐nm IMPs, which are distributed randomly in the membrane, and the subunits of OAPs (OAP‐Su, also 7 nm in diameter). Using cultured astroglial cells treated with basic fibroblast growth factor (bFGF), arginine vasopressin, or sorbitol, good evidence for a relationship between the density of 7‐nm IMPs and the size of OAPs can be demonstrated. These findings led to a hypothetical model of OAP modulation. A preliminary report has been published elsewhere (Neuhaus and Wolburg, 1989).
Glial response to axonal injury: In vitro manifestation and implication for regeneration
Authors: Sivron Tomer , Cohen Avi , Duvdevani Revital , Jeserich Gunnar , Schwartz Michal 
Published: January 2005

AbstractCrushed fish optic axons readily regenerate, while similarly injured rat optic axons do not; the reasons for the differences in regeneration ability may lie in differences in the environment of the axons. We have cultured glial cells from previously crushed optic nerves of fish and rat to determine whether a relationship exists between the ability to regenerate and the nature of the responses of the associated nonneuronal cells to injury. The glial cells were examined using indirect immunofluorescence with antibodies to known glial markers. In the rat cultures, mature GalC oligodendrocytes, which are known to be nonpermissive for axonal growth, were abundant. In contrast, in the fish cultures mature oligodendrocytes were rare, but A2B5positive cells were abundant. The high number of A2B5positive cells in the fish may suggest a high number of immature cells. This interpretation, however, should wait until evidence for glial cell lineage of the fish is available. Additional indication is provided also in the present study that the number of mature oligodendrocytes in the fish is regulated by elements external to the nerve. This study thus demonstrates an important difference between rat and fish optic nerves in the response of glial cells to the optic nerve injury.
Sodium channel expression detected with antibody 7493 in A2B5+ and A2B5− astrocytes from rat optic nerve in vitro
Authors: Minturn J. E. , Black J. A. , Angelides K. J. , Waxman S. G. 
Published: January 2005

AbstractAstrocytes cultured from neonatal rat optic nerve can be classified into two subtypes, distinguished by their morphology (stellate or fibroblast‐like) and their ability to bind monoclonal antibody A2B5. The presence of sodium channels in astrocytes cultured from rat optic nerve was demonstrated by indirect immunofluorescence with polyclonal antibody 7493, which is directed against purified rat brain sodium channel protein. Astrocytes cultured from postnatal day 7 (P7) rat optic nerves exhibited sodium channel immunostaining on both A2B5+ and A2B5− astrocytes up to 6 days in vitro (DIV). Staining was distributed throughout the cytoplasm and cell processes, with areas of greater intensity in the perinuclear region. At 6 DIV, the A2B5−/GFAP+ cells exhibited a loss of sodium channel immunostaining, while the A2B5+/GFAP+ cells continued to display 7493 immunoreactivity. This sodium channel staining pattern persisted for up to 28 DIV (the longest time point examined). Astrocyte cultures derived from P0 rat optic nerves exhibited sodium channel immunoreactivity during the first 6 DIV. The P0 astrocyte cultures, in which the vast majority of cells are A2B5−/GFAP+, displayed a similar staining pattern to those astrocytes with corresponding phenotype derived from P7 optic nerves. PO‐derived A2B5− astrocytes showed loss of 7493 immunostaining at 6 DIV, while the rare (< 1% of cells) A2B5+/GFAP+ cells continued to express sodium channels reactive to 7493. The reduction of sodium channel immunoreactivity in A2B5−, but not A2B5+, astrocytes from both P0 and P7 optic nerves after a similar latency (<6 DIV) suggests that the loss of immunostaining may result from the absence of neuronal associations in the culture environment, rather than an intrinsic biologically timed change in astrocytic expression of sodium channels.
Control of astrocyte volume by intracellular and extracellular Ca2+
Authors: Olson James E. , Fleischhacker Deborah , Murray W. Bruce , Holtzman David 
Published: January 2005

AbstractAstrocytes from primary culture were exposed to conditions that affect intracellular and extracellular Ca2+ concentrations. Astrocyte cell volume was increased approximately 16% after a 30 min exposure to isoosmotic phosphate‐buffered saline (PBS) containing the Ca2+ buffer EDTA. Cell volume returned to control values within 30 min of resuspension in normal PBS. Cellular calcium content was not affected by these treatments; however, the recovery of normal cell volume following EDTA exposure was inhibited by 0.1–1.0 mM quinine HCl in a dose‐dependent fashion suggesting that a potassium channel controlled by the intracellular Ca2+ concentration is important in this volume response. Intracellular accumulation of an exogenous Ca2+ buffer, BAPTA, also produced cell swelling that persisted following resuspension in normal PBS. Lowering the extracellular Ca2+ concentration with EDTA enhanced the swelling of BAPTA‐loaded cells. These data suggest that conditions leading to a decrease in free intracellular Ca2+ concentration may influence astrocyte volume by a mechanism similar to that described in other cell types.
Volume‐sensitive release of taurine from cultured astrocytes: Properties and mechanism
Authors: Pasantes‐Morale H. , Moran J. , Schousboe A. 
Published: January 2005

AbstractRelease of taurine in response to cell swelling induced by hyposmolarity was observed in cultured astrocytes. Efflux of 3H‐taurine increased by 30% and 70% upon reductions in osmolarity of only 5% and 10%. Reductions in osmolarity of 20%, 30%, and 50% stimulated basal taurine release by 300%, 500%, and 1,500%, respectively. The properties of this volume‐sensitive release of taurine were examined to investigate: 1) its association with K+ and Cl− fluxes, currently activated during volume regulation; 2) its relationship with Ca2+‐dependent reactions; and 3) the mechanism of the taurine efflux process.Taurine release was unaffected by removal of Na+, Ca2+, or Cl−, by pimozide and trifluoperazine, or by agents disrupting the cytoskeleton. The K+ channel inhibitors barium, quinidine, tetraethylammonium, and gadolinium had no effect. Taurine release was reduced by furosemide, a blocker of K+/Cl− cotransport, but not by the more specific inhibitor, bumetanide. It was markedly reduced by the inhibitors of Cl− channels DIDS, SITS, and anthracene‐9‐carboxylate. Taurine efflux was pH‐dependent, being reduced at low pH values. It was decreased at 4°C but not at 14°C or 20°C. These results suggest that the volume‐sensitive release of taurine is independent of K+ fluxes but may be associated with Cl− conductances. It also seems unrelated to Ca2+‐dependent transduction mechanisms. The Na+‐dependent taurine carrier apparently is not involved in the swelling‐induced release process.
Synthesis and phosphorylation of the glial fibrillary acidic protein during brain development: A tissue slice study
Authors: Noetzel Michael J. 
Published: January 2005

AbstractBrain slices were incubated with either [3H] amino acids or [32P] orthophosphate in order to characterize the synthesis and phosphorylation of the glial fibrillary acidic protein (GFAP) in the rat nervous system. The incorporation of [3H] amino acids into GFAP was found to increase significantly during early postnatal development, reaching a peak of activity on day 5 of life and then declining over the next 2 weeks. Concomitant with this peak of synthetic activity the content of GFAP in rat brain was also observed to increase dramatically. GFAP continued to accumulate in brain through postnatal day 30 despite a decrease in the synthesis of the protein. These results indicate that the increase in GFAP during the first month of life cannot be ascribed solely to the rate of GFAP synthesis. The findings are consistent with the hypothesis that during later stages of astrocytic development the accumulation of GFAP may be primarily dependent upon a low rate of protein degradation. The pattern of GFAP phosphorylation in the developing rat brain differed from that observed for the incorporation of [3H] amino acids. The peak incorporation of 32P into GFAP occurred on postnatal day 10 at a time when synthesis of the protein had declined by 43%. These findings suggest that during development phosphorylation of GFAP is mediated by factors different from those directing its synthesis. In addition, phsophorylation of GFAP did not alter its solubility in cytoskeletal preparations indicating that GFAP phosphorylation is probably not a major regulatory mechanism in disassembly of the astroglial filaments.