bluuebunny/crossref_metadata_2025_split huggingface dataset:
Establishment of a permanent rat brain‐derived glial cell line as a source of purified oligodendrocyte‐type 2 astrocyte lineage cell populations
Authors: Aloisi Francesca , Sun D. , Levi G. , Wekerle H. 
Published: January 2005

AbstractA permanent glial cell line (L3) has been established from mixed glial cultures obtained from neonatal rat forebrain by repetitive passaging and selection of the process‐bearing cells growing on top of a flat cell monolayer. Continuous propagation of the process‐bearing cells was supported by the flat cells, of presumed astroglial origin, which were present in negligible amounts following each passage but then grew and formed a basal, feeder layer. Throughout a culture period of over 2 years, the L3 cells have maintained a stable morphological and antigenic phenotype. In serum‐containing culture medium, most of the process‐bearing cells expressed at the same time features of immature oligodendrocytes (O4 positivity) and of astrocytes [glial fibrillary acidic protein (GFAP) positivity]. A smaller proportion of them was labeled by the monoclonal antibody LB1. LB1 + or O4+ cells were rarely GFAP−, and GFAP+ cells were rarely LB1− or O4−. GalC+ oligodendrocytes were seen only occasionally, but the proportion of these cells increased up to 30% upon culturing in chemically defined medium containing 0.5% fetal calf serum. The L3 process‐bearing cells accumulated the neurotransmitter γ‐aminobutyric acid (GABA), expressed the proteoglycan chondroitin sulfate, and responded to the mitogenic action of platelet‐derived growth factor (PDGF) and fibroblast growth factor (FGF). All these properties are characteristic of cells belonging to the 0‐2A (oligodendrocyte‐type 2 astrocyte) cell lineage. The L3 flat cells were largely negative for the glial markers tested, but resembled type 1 astrocytes in their ability to support the growth of 0‐2A lineage cells. The L3 cell line represents a unique source of large numbers of purified 0‐2A‐like lineage cells and may therefore represent a useful experimental system to analyze the functional and developmental properties of this glial population.
Chemotaxis by a CNS macrophage, the microglia
Authors: Yao J. , Harvath L. , Gilbert D. L. , Colton C. A. 
Published: January 2005

AbstractMicroglia demonstrate many characteristics similar to those seen in monocytes and tissue‐specific macrophages, including phagocytosis, production of oxygen radicals, and growth factors and expression of MHC antigens. We have examined the ability of microglia, cultured from the cerebral cortices of neonatal rats, to demonstrate another important functional characteristic of monocytic‐derived cells, that is, chemotaxis. Our results show that cultured rat microglia demonstrate chemotaxis to complement dependent chemoattractants such as recombinant C5a, zymosan activated serum, and to rat serum as well as to transforming growth factor‐beta, a chemoattractant produced by platelets, Microglia fail to migrate to bacterial dependent chemoattractants such as the N‐formyl peptides. The failure to respond is not dependenton maturational state of the microglia. Treatment with DMSO or casein, agents known to induce morphological and functional changes in cultured microglia reminescent of a “resting” and an “activated” macrophage, respectively, do not alter the response to fMet‐Leu‐Phe. In addition, the chemotactic response to serum in DMSO or casein‐treated cells is the same as the response seen in untreated day 10 cultured microglia or untreated age‐matched controls. The ability of microglia to migrate in response to inflammatory mediators suggests that these cells can move to sites of injury, thereby enabling them to participate in an inflammatory response.
Differentiation of rat hippocampal neurons induced by estrogen in vitro: Effects on neuritogenesis and Na,K‐ATPase activity
Authors: Blanco G. , Diaz H. , Carrer H. F. , Beaugé L. 
Published: January 2005

AbstractTo gain insight into the mechanisms responsible for differentiation of hippocampal neurons growing in vitro, the effects of estrogen on neuritic development and on activity and distribution of isoforms of the Na,K‐ATPase, were evaluated. Dissociated cells from hippocampi of 19‐day‐old rat fetuses were raised for 5 days in the presence or absence of 100 nM estradiol‐17β (E2) in minimum essential medium supplemented either with 10% untreated fetal calf serum (MEM‐10) or with 10% fetal calf serum previously adsorbed with dextran‐activated charcoal (MEM‐10‐Cha). Cultures in MEM‐10 showed larger neuritic length and increased levels of Na,K‐ATPase activity than cultures in MEM‐10‐Cha. In cells cultured in MEM‐10 medium, the addition of E2 resulted in selective enhancement of axonal length with a concomitant increase in the alpha‐2 isoform of the Na,K‐ATPase, whereas a decrease was found in the form most sensitive to ouabain; the total enzymatic activity remained unchanged. Conversely, in cultures raised in MEM‐10‐Cha, E2 did not affect Na,K‐ATPase activity or neuritogenesis. These results show presumably independent probes of cellular differentiation of hippocampal neurons (i.e., neuritogenesis and patterns of Na,K‐ATPase activity) were concurrently regulated by E2 and that such regulation depended on interaction with factor(s) present in calf serum. The well‐known neuritogenic effect of E2 is hereby extended to hippocampal neurons. although for these cells it seems to be restricted to axons.
Development of cholinergic markers in the neuroblastoma, N1E‐115
Authors: Kumar U. , Sellström Å. 
Published: January 2005

AbstractThe neuroblastoma, N1E‐115, was grown for 9 days after subcultivation. The development of acetylcholinestrase (AChE), choline acetyltransferase (CAT), QNB‐binding, choline uptake, and acetylcholine release was measured on days 1, 3, 6, and 9. In paralell experiments the irreversible AChE inhibitor soman was added to neuroblastoma on day 1 and the development of the above parameters except for release, was followed.AChE activity in the normal cells was found to develop also after confluency, whereas CAT activity and QNB‐binding followed the development of most of the cellular proteins, i.e., ceased to develop at confluency. Both choline uptake and acetylcholine release appeared independent of cellular development.In the soman‐treated cells the development of AChE was inhibited for up to 6 days and thereafter developed with the sama rate as in the normal cells. CAT and QNB‐binding developed as in the normal cells, but at a significantly reduced level. The development of choline uptake was not significantly different in soman‐treated and normal neuroblastoma.It is concluded that the development of the cholinergic marker QNB‐binding is intimately associated with that of the “presynaptic” marker CAT, whereas the development of AChE seems to be unrelated to these cholinergic parameters. The choline transport and the acetylcholine release seem to be equally well expressed on all the days studied.
Expression of myelin protein gene transcripts by schwann cells of regenerating nerve
Authors: Mitchell L. S. , Griffiths I. R. , Morrison S. , Barrie J. A. , Kirkham D. , McPhilemy K. 
Published: January 2005

AbstractThe expression of many myelin‐specific molecules in Schwann cells is profoundly decreased following denervation. This study examines the early reexpression of myelin protein genes associated with reinnervation. Following sciatic nerve crush, the distal, regenerated nerve was divided into appropriate (2.5 or 5 mm) consecutive lengths in which gene expression was monitored using Northern blotting, in situ hybridization, and immunostaining. The spatial separation of the distal axon tip and the more proximally located Schwann cells showing initial upregulation of Po mRNA was constant over the period of 5–13 days after crush at approximately 3–4 mm in fixed, processed material. Axons associated with Schwann cells showing the initial upregulation were completely or partially enveloped in Schwann cell cytoplasm, with very few having any degree of ensheathment. It is probable that only a limited axon‐Schwann cell contact is required for induction of the myelin protein genes. Myelin‐associated glycoprotein mRNA was upregulated prior to those for Po and myelin basic protein which had similar time courses. Reexpression of galactocerebroside also preceded that for Po mRNA. Signal abundance for all myelin proteins decreased in a proximal to distal direction from the crush site, and with time the “wave” of upregulation moved distally down the nerve. In the more proximal, remyelinating zones, the signal intensity exceeded that of the contralateral normal nerve. Signal intensity also varied considerably between adjacent, expressing Schwann cells. The data provide further evidence of the strong temporospatial relationship between axons and the regulation of myelin protein genes in Schwann cells.
Isolation of cDNAs from a mouse astroglial cell line by a subtracted cDNA library
Authors: Rhyner T. A. , Lecain E. , Mallet J. , Pessac B. 
Published: January 2005

AbstractAstrocytes belong to the glial cell population and represent a major subclass of the CNS. Although different subtypes of astrocytes have been described according to their morphological characteristics, biochemical markers of each subtype of astrocytes are not yet available. We have thus undertaken to compare gene expression pattern of different astroglial subtypes. In this study we have taken advantage of two astroglial cell clones derived from 8 day postnatal mouse cerebellar explants and which might be the in vitro equivalents of the velate protoplasmic (D19) and of the Golgi‐Bergmann (C8S) astrocytes (Alliot and Pessac, Brain Res, 306: 283–291, 1984). We have constructed a subtracted cDNA library derived from cytoplasmic poly(A)+ RNAs of the D19 cell line. This library was enriched 12‐fold for D19 specific sequences by subtractive hybridization with an excess of cytoplasmic poly(A)+ RNAs purified from the C8S astroglial clone. This subtracted library was differentially screened with cDNA probes derived from D19 and C8S cell lines; both probes were subtracted with C8S poly(A)+ RNAs. Eight cDNA clones corresponding to transcripts overexpressed in D19 were selected. Three cDNAs encode for smooth muscle actin, one for fibronectin and one for polyadenylate binding protein. The three other gene products have not been previously reported. The in vivo distribution pattern of these sequences in various mouse adult tissues shows that all these transcripts are expressed in the cerebellum and/or in the brain.
Characterization of nerve growth factor binding to embryonic rat spinal cord neurons
Authors: Marchetti D. , McManaman J. L. 
Published: January 2005

AbstractThe binding of iodinated β‐nerve growth factor, [125I]‐NGF, to embryonic (E16) rat spinal cord cells, was investigated to characterize the binding properties and cellular distribution of nerve growth factor receptors. Spinal cord cells prepared without trypsin yielded two classes of NGF binding sites with Kd's of 3 × 10−11 M and 4 × 10−9 M. Fractionation of the cells by discontinuous gradients composed of 8%, 12%, and 17% metrizamide was used to separate motoneurons from other cell types. The motoneuron enriched fraction (8% metrizamide) contained approximately 10% of the cells and 64% of the choline acetyltransferase (ChAT) activity. In contrast, the 12% metrizamide fraction contained most (51%) of the cells and 36% of the ChAT activity, while the 17% metrizamide fraction contained the remainder of the cells and negligible amounts of ChAT activity. Characterization of [125I]‐NGF binding to each metrizamide fraction showed that the motoneuron‐en‐riched fraction exhibited both high and low affinity binding sites, while the other metrizamide fractions exhibited only the low affinity binding sites. These findings indicate that although low affinity NGF receptors appear to be relatively evenly distributed amongst embryonic rat spinal cord cells, high affinity NGF receptors are found primarily on motoneurons.
Effects of in utero ethanol exposure on the developing dopaminergic system in rats
Authors: Druse M. J. , Tajuddin N. , Kuo A. , Connerty M. 
Published: January 2005

AbstractPrevious studies from this and other laboratories suggest that dopamine is decreased in selected brain regions of postnatal rats exposed to ethanol in utero. The present study expands previous work by examining the effects of in utero ethanol exposure on dopamine D1 and D2 binding sites and dopamine uptake in postnatal rats. In addition, dopamine content in the brain stem and frontal cortex of fetal and neonatal rats was examined.The experimental results indicate that in utero ethanol exposure markedly affects the postnatal development of the dopaminergic system in the striatum and frontal cortex. We observed a marked, transient deficiency of striatal dopamine (>40% decrease at 19 days) and dopamine uptake sites (∼ 25% decrease in V max at 35 days). The Bmax for striatal dopamine D1 binding sites was decreased by > 20% at both 19 and 35 days. Cortical D1 sites were markedly decreased at 19 days (> 40%). In contrast, the number of striatal D2 receptors was unaffected by in utero ethanol exposure at both ages.Analysis of tissue from neonatal rats demonstrated a marked dopamine deficiency in ethanol‐exposed rats on postnatal day 5. In light of the proposed morphogenic actions of dopamine early in development, it is possible that the early dopamine deficiency contributes to the abnormal postnatal development of the dopaminergic system.
Cholesterol esterification and Niemann–Pick disease: An approach to identifying the defect in fibroblasts
Authors: Bowler L. M. , Shankaran R. , Das I. , Callahan J. W. 
Published: January 2005

AbstractFibroblasts from 13 patients with the clinical phenotype of type IIS, Niemann–Pick disease were evaluated for their ability to incorporate oleic acid into cholesterol esters via an LDL responsive mechanism. Eight patients displayed a severe deficiency (< 8% of normal) of cholesterol ester (CE) synthesis while a clinically less affected group displayed intermediate levels (36% of normal) of synthetic capacity with no detectable overlap between these groups and the control range. There was no deficiency in cholesterol ester production in fibroblasts from a patient with Zellweger's disease, a disorder characterized by altered peroxisomes and abnormal peroxisomal cholesterol metabolism, while in I‐cell disease, characterized by a primary deficiency of a phosphotransferase which results in altered targeting of lysosomal hydrolases, it was reduced to 25% of the control level. To further implicate lysosomal proteins in the etiology of type IIS, Niemann–Pick disease we measured the effect of correction (conditioned) medium, and the lysosomotropic agent, NH4Cl on cholesterol ester synthesis in fibroblasts. NH4Cl completely inhibited incorporation into CE by normal cells, thus mimicking the CE defect in type IIS, Niemann–Pick cells. Conditioned medium had no effect on incorporation into CE synthesis but medium conditioned in the presence of 10 mM NH4Cl stimulated incorporation into CE in the control but not in Niemann–Pick cells. When Niemann–Pick cells cultured in the presence of NH4Cl were challenged to synthesize CE in the absence of NH4Cl, a significant enhancement of CE synthesis was noted in representative cell lines from both groups of patients. These results indicate that two levels of deficiency can be clearly identified and that this biochemical phenotype appears to correlate with the severity of clinical disease. We suggest that the defect in patients with intermediate levels of CE synthesis may reside in a phosphomannosyl‐directed glycoprotein that is targeted to the lysosome via the phosphomannosyl receptor, while the most severely affected patients may be defective in another type of protein.
Cortical glucose metabolism in parkinson's and alzheimer's disease
Authors: Peppard R. F. , Martin W. R. W. , Clark C. M. , Carr G. D. , McGeer P. L. , Calne D. B. 
Published: January 2005

AbstractCharacteristic regional patterns of decreased cerebral glucose metabolism (rCMRG) have been described in a variety of neurodegenerative conditions associated with dementia. The present study was undertaken to determine whether the metabolic pattern in Parkinson's disease is altered by the presence of impaired cognitive function. Glucose metabolism was measured with positron emission tomography in 6 patients with Parkinson's disease and dementia (PDD), 8 patients with Parkinson's disease and normal cognition (PD), and 6 patients with Alzheimer's disease (AD). All AD patients subsequently had the diagnosis proven neuropathologically at autopsy. Correlation coefficients of the metabolic rates across 32 regions of interest were calculated between each pair of patients. Q‐component analysis of the correlation matrix showed that the AD and PD groups formed two distinct clusters and that the PDD group had a metabolic pattern which was similar to that of the AD group. Comparison of standardized rCMRG values showed that the PDD group differed from the PD group in having significantly lower relative rCMRG in the left perirolandic and bilateral angular gyrus regions. There were no significant differences between the PDD and AD groups. These results suggest a similar pattern of cortical dysfunction in both Alzheimer's disease and in Parkinson's disease/dementia.
Recombinant human β‐nerve growth factor (NGF): Biological activity and properties in an enzyme immunoassay
Authors: Söderström S. , Hallböök F. , Ibáñez C. F. , Persson H. , Ebendal T. 
Published: January 2005

AbstractNerve growth factor (NGF) supports sympathetic and sensory neurons in the peripheral nervous system and also functions in the development and maintenance of cholinergic neurons in the basal forebrain. NGF distribution can be studied in the brain of the rat and mouse with the use of a sensitive two‐site enzyme immunoassay (EIA) for mouse NGF. It would be of interest to measure the NGF protein also in the human brain, especially against the background that the cholinergic neurons are severely deteriorated in senile dementia of the Alzheimer type. The limited immunological cross‐reactivity between NGFS from different species has previously hampered attempts to determine levels of the human NGF. We have now examined the biological activity and immunological properties of human recombinant NGF protein in medium conditioned by COS cells transfected with the human NGF gene. The human NGF behaved similar to mouse NGF in a sympathetic ganglion bioassay. The monoclonal antibody 27/21 to mouse NGF was shown to effectively block the activity of both the human recombinant NGF and mouse native NGF. A two‐site EIA using monoclonal antibody 27/21 was optimized. Under the conditions used, the EIA detected the human recombinant NGF with the same sensitivity (1 pg/ml) as shown for the mouse NGF. It should now be possible to test this EIA also on homogenized tissue to examine human NGF in brain samples from Alzheimer patients and age‐matched controls.
Basic fibroblast growth factor: A potential autocrine regulator of human glioma cell growth
Authors: Gross J. L. , Morrison R. S. , Eidsvoog K. , Herblin W. F. , Kornblith P. L. , Dexter D. L. 
Published: January 2005

AbstractBasic fibroblast growth factor (bFGF) is a potent mitogen and angiogenic factor. bFGF is expressed by a variety of solid human tumors and has been implicated as an autocrine regulator of tumor growth. Different solid tumor lines including glioma, colon carcinoma and melanoma were examined for intracellular immunoreactive bFGF, high‐ and low‐affinity bFGF receptors and mitogenic response to bFGF when grown in chemically defined medium. All tumor lines contained significant levels of bFGF. In addition, all tumor lines contained subsets of five forms of inmiunoreactive bFGF, as well as 0.68–20 × 106 low affinity bFGF binding sites (Kd = 15–300 nM). Most, but not all lines exhibited high affinity bFGF receptors (Kd = 25–40 pM). Glioma cell lines were distinguished by expressing the highest levels of bFGF protein as well as the most high‐affinity receptors for bFGF. Furthermore, glioma cell lines were the only tumor type mitogenically responsive to bFGF. These results indicate that glioma cells express high levels of this potent mitogen and angiogenic factor relative to human colon carcinoma and melanoma cells. The expression of bFGF and bFGF receptors by glioma cells may be related to abnormal growth and neoplastic progression in these tumors.
Single K+ channel properties in cultured mouse Schwann cells: Conductance and kinetics
Authors: Verkhratsky A. , Hoppe D. , Kettenmann H. 
Published: January 2005

AbstractCultured Schwann cells are characterized by a strong outward rectification of the membrane; the threshold of the outward currents is close to the resting membrane potential of about ‐50 mV (Gray et al.: In Ritchie, Keynes (eds): Ion Channels in Neural Membranes. New York: Alan R. Liss, Inc., pp 145–157, 1986). These outward currents show up a heterogeneity among the cultured Schwann cells: some cells displayed inactivating, others non‐inactivating outward currents (Hoppe et al.: Pflügers Arch 415:22–28, 1989). In this study we characterized the single channel currents using the patch‐clamp technique in the intact patch recording configuration. The conductance of all recorded channels was 10‐12 pS. (5.6 mM [K+]o) These channels were K+ selective since changes in extracellular [K+] resulted in changes of the reversal potentiaJLas predicted for an exclusively K+ selective pore. The reversal potentials also predicted an intracellular [K+] of 60 mM indicating that the K+ equilibrium potential is slightly negative to the membrane potential. Analysis of the kinetic behavior of the channels resolved two different types of behaviour:40% inactivated during a depolarizing voltage step, the others showing no sign of inactivation. The analysis of open probability and gating properties in the steady state showed up more differences between these two channel types: mean open probability peaked at about 10 mV for inactivating channels, while it continuously increased for non‐inaclivating channels. The inactivation time constants of averaged single channel and whole cell currents were similar and showed both a similar voltage dependency. We conclude that cultured. Schwann cells express either two types of K+ channels with Slimlar conductance or a channel which can acquire two functional states and that these channels can account for the different types of K+ currents observed in these cells.
Lithium chloride alters cytoskeletal organization in growing, but not mature, cultured chick sensory neurons
Authors: Hollander B. A. , Bennett G. S. 
Published: January 2005

AbstractBiochemical analysis indicates that lithium ion (Li+) has deleterious effects on the metabolism of at least two elements of the cytoskeleton in cultured chick dorsal root ganglia (DRG) neurons. Phosphorylation ofnewly synthesized middle molecular mass neurofilament polypeptide (NF‐M) is inhibited by 10‐25 mM LiCl, and tubulin (Tb) synthesized in the presence of Li+ is subject to rapid degradation. These Li+‐in duced metabolic abnormalities are accompanied by alterations in cellular and cytoskeletal morphology. Treatment of cultures having vigorously growing neurites with 25 mM LiCl results in the cessation of net neurite growth, without causing neurite retrac tion. Indirect immunofluorescence reveals that in these cultures Li+ provokes an aggregation of NF protein into a dense knot in the cell body/proximal neurite region. The knots contain accumulations of all three NF polypeptides and electron microscopic observation demonstrates that the knots contain in tact, but disorganized, filaments. Both the inhibition of neurite outgrowth and NF collapse are reversible. Tubulin and intact microtubules are redistributed in immature cultures treated with Li+ insofar as they are excluded from the NF knots. Neurons in estab lished cultures (e.g., 7 days and beyond) fail to show any difference between Li+ treatment and control conditions in the morphology of the cytoskeletal ele ments examined.