bluuebunny/crossref_metadata_2025_split huggingface dataset:
Establishment, characterization, and evolution of cultures enriched in type‐2 astrocytes
Authors: Aloisi F. , Agresti C. , Levi Giulio 
Published: January 2005

AbstractThe aim of the present study was to prepare cultures enriched in type‐2 astrocytes (AS) and to analyze some of the properties of these cells over relatively long culture periods. Cultures enriched in type‐2 AS were obtained by subculturing, at low cell density and in the presence of fetal calf serum, a cell population containing numerous bipotential glial precursors. This cell population was detached mechanically from 2‐ to 3‐week primary mixed glial cultures prepared from 1‐day postnatal rat cerebral cortex. The cellular composition of the subcultures was analyzed immunocytochemically over a period of 3 weeks using various combinations of antibodies, recognizing a set of differentiated and a set of undifferentiated glial antigens (glial fibrillary acidic protein [GFAP], galactocerebroside, sulfatide, gangliosides binding the monoclonal antibodies A2B5 and LB1, fibronectin). Most LB1+, A2B5+ glial precursors differentiated into type‐2 AS within a week. At this stage, type‐2 AS accounted for more than 70% of cells in the cultures and exhibited the characteristic features previously described for these cells (stellate shape, GFAP, LB1 and A2B5 positivity, ability to accumulate [3H]GABA and to synthesize chondroitin sulfate, low proliferative activity). About one third of the type‐2 AS also were recognized by O4 (antisulfatide) antibodies. The major contaminants were macrophages (10–15%) and fibroblastic cells (5–10%). In longer term cultures, type‐2 AS tended to lose several of these features. Many acquired a flat, polygonal shape and lost LB1 positivity. The ability to accumulate [3H]GABA progressively decreased, as did the expression of chondroitin sulfate, although to a lesser degree. Although losing several of their properties, type‐2 AS did not appear to acquire the properties of type‐1 AS: their proliferative activity remained very low, and they did not express class II antigens of the major histocompatibility complex upon stimulation with γ‐interferon. Some became positive for fibronectin.
Growth cone migration across extracellular matrix components depends on integrin, but migration across glioma cells does not
Authors: Letourneau P. C. , Pech I. V. , Rogers S. L. , Palm S. L. , McCarthy J. B. , Furcht L. T. 
Published: January 2005

AbstractTo promote neurite elongation, nerve growth cones must adhere to other surfaces. A complex of integral membrane glycoproteins mediates cell binding to the extracellular glycoproteins fibronectin and laminin (Horwitz et al., J Cell Biol 101:2134–2144, 1985). The receptor complex, named integrin, binds to fibronectin by recognition of a specific peptide sequence, Arg‐Gly‐Asp‐Ser (RGDS), in the fibronectin molecule (Pierschbacher and Ruoslahti, Proc Natl Acad Sci USA 81:5985–5988, 1984). We have used antibodies to integrin and an RGDS synthetic peptide to probe the functions of integrin in the migration of growth cones extended from sensory and spinal cord neurons of chick embryos. Analyses of time lapse videotapes of growth cone migration before and after adding RGDS indicated that 2 mM RGDS rapidly inhibits growth cone movement on substrata coated with fibronectin or a fragment of fibronectin containing the RGDS sequence. RGDS has no effect on growth cone movement on laminin or on a surface coated with material deposited from heart conditioned medium. However, a monoclonal antibody to the integrin complex (10 μg/ml CSAT) completely blocks growth cone movement on substrata treated with fibronectin, laminin, or heart conditioned medium. Thus integrin may be involved in growth cone adhesion to several extracellular molecules, although the selective effects of RGDS indicate that the integrin complex may have heterogeneous sites for interaction with different components of the extracellular matrix. CSAT antibody has no discernible effect, however, on growth cone migration across the upper surfaces of C6 glioma cells. These data indicate that the surfaces of nerve growth cones contain multiple binding molecules that mediate different adhesive interactions during migration.
Development of cholinergic pedunculopontine neurons in vitro: Comparison with Cholinergic septal cells and response to nerve growth factor, ciliary neuronotrophic factor, and retinoic acid
Authors: Knusel B. , Hefti F. 
Published: January 2005

AbstractThe well‐documented role of nerve growth factor (NGF) in the function of cholinergic neurons in the mammalian basal forebrain can be regarded as a paradigm for the action of trophic substances on CNS neurons. Although several growth factors have been identified in recent years, the specificities and importance of such factors for the development of the nervous system are still unknown. In the present study it has been tested whether NGF affects the group of pedunculopontine cholinergic neurons. This population, which has been described in detail only recently, is located more caudally than but resembles, in some aspects, the basal forebrain cholinergic neurons. The cell bodies are located in the metencephalic pedunculopontine and dorsolateral tegmental nuclei. Similar to the forebrain cholinergic neurons, they are medium to large in size and ascend centrally with long axons. Projection areas are widespread throughout the mesencephalon and diencephalon. Dissociated pontine and septal cells of fetal rat brain (embryo ages E14 to E17) were grown in culture for 7 to 14 days in the presence or absence of NGF. Furthermore, a possible action of retinoic acid and ciliary neuronotrophic factor (CNTF) on cholinergic neurons of both the basal forebrain and the pontine area were tested. Differentiation of cultured cholinergic neurons was assessed by biochemical determination of choline acetyltransferase (ChAT) activity and by immunocytochemical staining for ChAT.NGF in concentrations of 1 to 1,000 ng/ml medium increased the number of immunostained cells and the staining intensity in ChAT immunocytochemistry and enhanced ChAT activity by at least 100% above control levels in septal cultures, thus confirming earlier results. In marked contrast, the same concentrations of NGF failed to influence ChAT activity or immunocytochemical staining in cultures of the pontine area. Retinoic acid (10−8 M to 10−5 M) and CNTF (0.2 and 2.0 ng/ml, corresponding to 1 and 10 trophic units, as defined in the ciliary ganglion cell assay) failed to enhance ChAT activity in either culture system and did not potentiate the NGF‐mediated increase of ChAT activity in septal cultures. Our results, which indicate that pedunculopontine cholinergic neurons do not respond to NGF during development, are in line with those of NGF‐receptor visualization studies that failed to demonstrate such receptors on cholinergic pontine cells in postnatal and adult rats. The findings further underline the specificity of NGF action in the central nervous system and, in particular, do not support the idea of transmitter‐specific neurotrophic factors. Neither NGF nor CNTF are general cholinergic growth or survival factors.
Projection‐neurons that send axons through the lumbar spinal cord of the chick embryo are not obviously distributed in a segmentally repetitive pattern
Authors: Schlosser G. , Tosney K. W. 
Published: January 2005

AbstractEarlier studies have shown that the formation of the segmentally repetitive pattern of spinal nerves in avian embryos depends upon the segmental arrangement of the somites and does not reflect an intrinsic pattern of the spinal cord. These studies could not, however, rule out the possibility that some elements of the central nervous system are intrinsically segmented. The question remained, for instance, whether projection‐neurons are distributed in a segmentally repetitive pattern within the spinal cord. To address this question, horseradish peroxidase was injected into one or two segments of the lumbar spinal cord of chick embryos, thereby labeling projection‐neurons that had ascending or descending axons passing through the injection site. In all stages examined, the number of labeled projection‐neurons in the anterior and posterior halves of each segment did not differ significantly. In addition, segmentally repetitive peaks or troughs in the numbers of labeled projection‐neurons were not detected in the center of each segment. Three subpopulations of projection‐neurons, defined by their position along the dorsal‐ventral axis of the spinal cord, were also not segmentally distributed by these criteria. While these results do not rule out the possibility that subpopulations of projection‐neurons defined by dendritic morphology, functional class, or some other parameter are segmentally arranged or that there is a basic modular repetition of neural populations from segment to segment, they do show that projection‐neurons as a class and subpopulations of these neurons defined by their dorsal‐ventral position are not obviously distributed in a repetitive segmental pattern.
Developmental regulation of protein tyrosine phosphorylation in rat brain
Authors: Aubry M. , Maness P. F. 
Published: January 2005

AbstractProteins phosphorylated at tyrosine residues in the developing rat brain have been identified with a focus on the nerve growth cone and synaptic terminal. Endogenous protein phosphorylation in membranes from a subcellular growth cone fraction of fetal rat brain revealed prominent 55–60 kD phosphotyrosine‐contaning proteins. Proteins of similar size were recognized by phosphotyrosine antibodies in isolated growth cone membranes, indicating that they contained phos photyrosine in vivo. Proteins of 55–60 kD were not highly phosphorylated in synaptosomes from adult brain, suggesting a growth cone‐specific function. Generally, tyrosine phosphorylation was much lower in adult brain than in fetal brain fractions. Although some synaptosomal membrane proteins that contained phosphotyrosine corresponded in size with those in growth cone membranes (92 kD, 41 kD), others were unique to synaptosomal membranes (38 kD and 30 kD).Immunoperoxidase staining of fetal rat neocortex with phosphotyrosine antibodies at embryonic day 19 revealed immunoreactivity in presumptive migratory neuroblasts in the intermediate zone and in processes of the molecular layer. Proliferating neuroepithelial cells of the ventricular zone showed little immunore‐activity. Lower levels of phosphotyrosine immunore activity were seen until postnatal day 10, correlating with the period of maximal process outgrowth. These results indicate that protein tyrosine phosphorylation in the developing nervous system may be functionally significant in an aspect of neuronal differentiation such as growth cone‐mediated process extension and cell migration. An analogous role in the mature brain may be related to synaptic plasticity or function.
Synthesis of chimeric mouse nerve growth factor precursor and human β‐nerve growth factor in Escherichia coli: Immunological properties
Authors: Dicou E. , Houlgatte R. , Lee J. , von Wilcken‐Bergmann B. 
Published: January 2005

AbstractThe complete mouse prepro‐nerve growth factor (NGF) DNA was fused to the carboxyl terminus of the β‐galactosidase (lac‐z) gene of Escherichia coli. Similarly, a genomic fragment encoding the human NGF comprising codons 11 to 106 (from a total of 118) was fused to the fifth codon of the amino terminus of β‐galactosidase. Both bacterial vectors produce high amounts of the chimeric proteins. After cell lysis most of the chimeric mouse preproNGF protein is insoluble and appears in the pellet, whereas the majority of the chimeric human β‐NGF remains in the supernatant.Purification of the fusion proteins from the soluble fraction was achieved by affinity chromatography to p‐aminophenyl β‐D‐thio‐galactoside Sepharose. Yields of the purified chimeric proteins were increased threefold to fourfold by the addition of protease inhibitors in the lysis and chromatography buffers. Their antigenic similarity to the preproNGF and mouse β‐NGF was examined by their interaction to sera raised against synthetic peptides which reproduce sequences of the precursor protein and to sera directed against native and denatured mouse β‐NGF using enzymelinked immunoabsorbent assay (ELISA) techniques. Antibodies to the peptide N2 (—163 to —139) interacted with high affinity with the chimeric mouse preproNGF protein. Antisera to native and denatured mouse β‐NGF interacted with both chimeric proteins but with a variable degree of affinity. These results provide direct evidence that certain antisera to mouse β‐NGF can cross‐react with the human β‐NGF molecule.
Novel astrocytic protein in multiple sclerosis plaques
Authors: Malhotra S. K. , Predy R. , Johnson E. S. , Singh R. , Leeuw K. 
Published: January 2005

AbstractMonoclonal antibody J1‐31 (MAb J1‐31, isotype IgG 2b) was raised against crude homogenate of brain tissue from a multiple sclerosis (MS) patient (autopsy sample; Malhotra et al.: Microbios Letters 26:151–157, 1984). In human brain, MAb J1‐31 recognizes an intracellular protein antigen (J1‐31 antigen), which bands at approximately 30,000 daltons under reducing conditions for sodium dodecyl sulfate gel electrophoresis (Singh et al.: Bioscience Reports 6:73‐79, 1986). By immunofluorescence microscopy, MAb J1‐31 stains those cells that are also stained by antiserum to glial fibrillary acidic protein (GFAP), namely astrocytes, retinal Müller cells, and tanycytes in the ependyma (Predy et al.: Bioscience Reports 7:491–502, 1987). In addition, MAb J1‐31 stains ciliated ependymal cells that do not express GFAP.Using a model system for gliosis (laceration‐type injury of rat spinal cord), we were able to show that astrocytes responding to central nervous system injury exhibit greatly enhanced staining for J1‐31 antigen (Predy et al.: Journal of Neuroscience Research 19:397–404, 1988; Predy and Malhotra: Brain Research Bulletin in press, 1989). In this article, we demonstrate that immunofluorescence staining owing to MAb J1‐31 is greatly enhanced in MS plaques, as compared to adjacent “apparently normal” white matter. (This is consistent with previous results as MS plaques characteristically show an astroglial response [reactive gliosis] leading to the formation of a glial scar [McKhann: Annual Review of Neuroscience 5:219–239, 1982].) In addition, we present further evidence that J1‐31 antigen is distinct from GFAP, although these two proteins may be associated spatially with one another.
Interaction of myelin basic protein and proteolipid protein
Authors: Edwards A. M. , Ross N. W. , Ulmer J. B. , Braun P. E. 
Published: January 2005

AbstractThe interaction of myelin basic protein (MBP) and proteolipid protein (PLP) was studied using a microtitre well binding assay and the ligand‐blot overlay technique. The binding of iodinated PLP to MBP that was immobilized on microtitre wells was saturable and reversible. Its selectivity was investigated by the ligand‐blot overlay technique. Iodinated PLP was found to bind MBP but not any other CNS myelin proteins. This interaction was not dependent on the phosphoryl moiety of MBP. Binding of PLP to histone H4 also occurred, but the amount of PLP bound per unit MBP was greater than for this histone.
Identity of ED2‐positive perivascular cells in rat brain
Authors: Graeber M. B. , Streit W. J. , Kreutzberg G. W. 
Published: January 2005

AbstractA controversial, though fundamental, issue in neurobiology concerns the nature, origin, and function of brain macrophages. By immunocytochemical analysis using monoclonal antibodies directed against rat macrophage antigens, i.e., ED1 −3, Ox‐41, Ox‐42, and KiM2R, we show that a group of perivascular cells located within the basal membrane of CNS blood vessels are immunoreactive. These cells, which resemble pericytes in terms of their anatomical distribution, are distinct from resting parenchymal microglia immunologically as well as morphologically. Our results demonstrate considerable heterogeneity in the immunophenotype of resident brain macrophages, which may be part of the immune—nervous system interface.
Human malignant astrocytoma xenografts migrate in rat brain: A model for central nervous system cancer research
Authors: Bernstein J. J. , Goldberg W. J. , Laws E. R. 
Published: January 2005

AbstractFresh cells from two grade 3 human malignant astrocytomas were prelabeled with Phaseolus vulgaris leucoagglutin (PHAL) and then xenografted into freshly made implantation pockets in rat host cerebral cortex. Animals were sacrificed at 7, 14, 21 days and 1 month postimplantation (DPI). Paraffin sections were double‐labeled for the presence of glial fibrillary acidic protein (GFAP), a specific marker for astrocytes and differentiated astrocytoma cells, and PHAL, utilized as a marker for graft‐derived cells. Grafted human astrocytoma cells were found on the glia limitans along the entire circumference of the brain, in the corpus callosum, internal capsule, entopeduncular nucleus, optic tract, and median eminence. In addition, astrocytoma cells were observed in the cingulum, habenula, arcuate, and supraoptic nucleus. Astrocytoma cells entered the spaces of Virchow‐Robin, and migrated along parenchymal blood vessels and between the ependymal and subependymal layers of the third and lateral ventricles. The corpus callosum was a major migration route for the astrocytoma cells. The presence of basal lamina or parallel nerve fiber bundles was a common factor for these migration routes. The migration of the human astrocytoma xenografted cells in the rat brain followed the spread of human malignant astrocytomas in the human brain and is a valuable basic science tool in brain cancer research.
Selectin of encephalitogenic rat T‐Lymphocyte clones recognizing an immunodominant epitope on myelin basic protein
Authors: Chou Y. K. , Vandenbark A. A. , Jones R. E. , Hashim G. , Offner H. 
Published: January 2005

AbstractUsing the soft agar‐cloning technique, we isolated 13 T‐cell clones from guinea pig basic protein (GP‐BP)‐specific T‐cell lines derived from Lewis rats. The clonal frequency was approximately 2.5 × 10−5. Each of these clones had a similar but not identical pattern of response to a battery of synthetic peptides representing overlapping epitopes in the encephalitogenic region for Lewis rats (69–89 sequence). All clones responded to the minimal encephalitogenic sequence (residues 72–84) restricted by I‐A but not I‐E molecules, and all transferred clinical experimental autoimmune encephalomyelitis (EAE) and delayed‐type hypersensitivity (DTH) reaction to naive rats. Phenotypically, the clones were W3/13+ (total T), W3/25+ (T helper), and OX‐22+ (DTH associated). This report demonstrates for the first time the applicability of the soft agar‐cloning technique for obtaining encephalitogenic T‐cell clones. The exclusive recovery of 72–84‐specific T‐cell clones after only two rounds of stimulation with GP‐BP indicates the immunodominance of this epitope and the power of the line selection technique for obtaining encephalitogenic T‐cell specificities.
Characterization of antibodies to synthetic nerve growth factor (NGF) and ProNGF peptides
Authors: Ebendal T. , Persson H. , Larhammar D. , Lundströmer K. , Olson L. 
Published: January 2005

AbstractSequence data for the mature nerve growth factor (NGF) protein and its precursor are available from molecular cloning of the NGF gene in several species, including mice, humans, rats, and chickens. Hydrophilicity analysis of the predicted rat and chicken prepro‐NGF was carried out to locate putative antigenic determinants. Eight peptides were selected and synthesized based on hydrophilicity profiles. Two peptides represent sequences in the rat (and mouse) pro‐NGF, one peptide (our peptide P3) represents a highly conserved region of the mature NGF protein (identical in humans, mice, rats, and chickens), two peptides are specific for the mature chicken NGF, and the remaining three peptides are specific for the mature rat NGF (each with only one amino acid substitution compared with corresponding segments of the mouse NGF). For immunization, the peptides were conjugated to keyhold limpet hemocyanin and used to produce antisera in rabbits. After bleeding, peptidespecific antibodies were purified on affinity columns prepared by coupling each of the synthetic peptides. The different peptide antisera and affinity‐purified antibodies then were characterized by enzyme‐linked immunoassay (ELISA) and immunohistochemistry of the male mouse submandibular gland, a rich exocrine source of NGF. ELISA analysis showed that all peptide antisera bound two to four orders of magnitude better than normal rabbit serum to a coat of their proper peptide. The higher binding was retained by the purified peptide antibodies compared with normal rabbit immunoglobulin. Specific tests, in which one peptide antiserum was checked against different peptide coats in the ELISA, also showed two to four orders of magnitude higher binding of antibodies to the proper synthetic peptide. The peptide antibodies also were tested for their ability to bind to native mouse βNGF coated to the immunoplates. Only antibodies raised to the conserved P3 peptide recognized native NGF to an extent similar to that obtained with polyclonal anti‐NGF antibodies. Conversely, P3 was well recognized by several different NGF antisera.Immunohistochemically, both peptide antisera against the pro‐NGF stained the perinuclear cytoplasm in the basal part of the cells of the granulated convoluted tubules in the mouse submandibular gland. Several of the peptide antisera against the mature rat NGF strongly stained the secretory granules in the same cells. It was concluded that the conserved P3 epitope of the mature βNGF is a region useful to raise peptide antibodies for recognition of the native NGF, possibly from a number of species, and that several of the antisera raised in this study have potential use for immunohistochemical localization of NGF and prepro‐NGF.
Detection and partial biochemical characterization of a novel 57,500 Dalton protein in rat brain myelin
Authors: Miller S. L. , Ötvös‐Papp E. , Prichett W. , Meyer R. D. 
Published: January 2005

AbstractUsing a monoclonal antibody (P6C3) derived from mice immunized with partially delipidated rat myelin, we detected a single 57.5 kDa protein species (BT57.5) positioned between α‐ and β‐tubulin on nitrocellulose containing electrophoretically separated rat brain myelin proteins. The kinetics of incorporation of 14C‐labeled BT 57.5 and myelin basic protein into myelin was similar in rats injected intracranially with [14C]amino acids to radioactively label these proteins. Immunoblotting of the separated proteins of the myelinlike fraction of rat brain with the anti‐BT57.5 monoclonal antibody occasionally revealed a faint band corresponding to BT57.5 but consistently showed a more prominent protein band that migrated slightly ahead of BT57.5. When the myelin and myelinlike fraction were isolated from rats injected with [14C] amino acids, labeling of the prominent anti‐BT57.5 binding protein in the myelinlike fraction preceded that of BT57.5 in myelin, reaching a peak of labeling and then decreasing before BT57.5 reached its peak level. Thus, the prominent anti‐BT57.5 protein in the myelinlike fraction might be a metabolic precursor of BT57.5 in myelin or the myelinlike fraction is simply turning over more rapidly and is distinct from the myelin fraction. BT57.5 was detectable in rat, mouse, guinea pig, bovine, and human CNS myelin but not rabbit and was detectable in the peripheral nervous system only in myelin of rats and humans. Finally, in extensively purified myelin, BT57.5 appeared to copurify with myelin basic protein, suggesting that BT57.5 is a constituent of myelin rather than an artifact arising during brain homogenization procedures.