bluuebunny/crossref_metadata_2025 huggingface dataset:
Expression of GD3 ganglioside by developing rat cerebellar purkinje cells in situ
Authors: Reynolds Richard , Wilkin G. P.
Published: January 2005

GD3 is a major ganglioside of the immature vertebrate CNS, and its expression is suggested to be characteristic of immature neuroectodermal cells. Using immunocytochemistry on cryostat sections of developing rat cerebellum with a monoclonal antibody specific for GD3, we have found that GD3 begins to be expressed on the plasma membrane of Purkinje cell bodies and dendrites beginning at postnatal day 7. Staining became brighter as the dendritic tree of the cells enlarged. As the Purkinje cells began to mature in different folia, they became GD3+, until by 15 days postnatal all Purkinje cells were GD3+. Positive staining of the dendritic tree was still present in the adult cerebellum. Using a monoclonal antibody 7–8D2, which recognizes cerebellar granule cells and their axons (the parallel fibres), and polyclonal antibodies against a synaptic vesicle component synaptophysin, double‐immunofluorescence staining together with anti‐GD3 antibodies suggested that the appearance of GD3 immunoreactivity did not correlate either with the ingrowth of parallel fibres or the presence of their synapses on Purkinje cell dendrites. However, comparison with earlier morphological studies showed that the appearance of GD3 immunoreactivity correlated well with the formation of climbing fibre synapses on Purkinje cell dendrites and the onset of the rapid expansion of the dendritic tree. These results are in keeping with the idea that elevated GD3 concentrations are found in certain cell types during periods of rapid growth or high metabolic activity but also show that this is not only restricted to immature cells.
Role of arachidonic acid and other free fatty acids in mitochondrial dysfunction in brain ischemia
Authors: Hillered Lars , Chan Pak H.
Published: January 2005

The aim of the present investigation was to evaluate the possible role of arachidonic acid and other free fatty acids in ischemia‐induced mitochondrial dysfunction. Respiratory activities were measured in mitochondria isolated from rat brains subjected to 15–30 min of decapitation ischemia. Addition of bovine serum albumin (BSA) to the mitochondria, isolated in BSA‐free media, abolished an ischemia‐induced increase in substrate‐stimulated (state 4) respiration but only partly reversed a marked inhibition of substrate‐, phosphate‐, and ADP‐stimulated (state 3) respiration caused by the ischemia. Individual free fatty acids were measured in aliquots of the same mitochondrial preparations before and after treatment with BSA. There was a significant increase in arachidonic (20:4), stearic (18:0), palmitic (16:0), and docosahexaenoic (22:6) acid during ischemia. BSA treatment removed all 20:4 and reduced the amount of 18:0 and 16:0, but had no significant effect on 22:6. The main conclusions were (1) that 20:4, 18:0, and 16.0 were responsible for the partial uncoupling (increase in state 4 respiration) of mitochondrial respiration during ischemia, (2) that the inhibition of state 3 respiration caused by ischemia could only partly be attributed to an effect of FFAs, and (3) that the ischemia‐induced mitochondrial dysfunction was caused by a combination of factors including 20:4.
Antigen recognized by monoclonal antibodies to mesencephalic neural crest and to ciliary ganglion neurons is involved in the high affinity choline uptake mechanism in these cells
Authors: Barald Kate F.
Published: January 2005

High‐affinity choline uptake mechanisms are among the characteristics of cholinergic neurons such as the ciliary and choroid subpopulations in the ciliary ganglion (Barald and Berg, 1979).

We have produced three monoclonal antibodies (Mabs), two of which were made to 8‐day embryonic chick ciliary ganglion (CG) neurons (CG‐1, CG‐4) (Barald, 1982) and one of which was made to cultured mesencephalic neural crest (NC) cells (CG‐14) removed from the embryo 31 hr after incubation. We have shown that all three Mabs label a common 75 kD antigen present on the cell surface of both CG neurons and NC cells (Barald, 1988).

Here we report that the CG‐1 and CG‐4 antibodies, used in the same ratios in which they are synergistically cytotoxic for both the CG and NC cells (Barald, 1988), and Mab CG‐14 alone, have specific effects on the high‐affinity choline uptake mechanism (HACU) of CG neurons and isolated antigen‐positive NC cells in the absence of complement. CG‐1 and CG‐4 in ratios of 8/1 (the same ratios that are used to kill the CG and the NC subpopulation), but neither singly, inhibit the HACU of CG neurons by 40% and that of isolated antigen‐positive NC cells by 75%. However, CG‐14 alone, at 1 μg/ml, inhibits the HACU of both CG neurons and isolated NC cells by 95%.

None of the antibodies had an effect on numbers of ouabain binding sites (a measure of the Na+/K+ ATPase) or cell surface acetylcholinesterase (AChE) of CG neurons or NC cells isolated by “no‐flow” fluorescence cytometry with a Meridian Instruments ACAS470 cytometer. CG or NC cells grown in the presence of the antibodies without complement grow and remain healthy for many weeks. They exhibit no difference in morphology, protein content, lactate dehydrogenase activity (LDH), or division time from untreated sister cultures.

Therefore, the antigen recognized by all three Mabs may be involved in a high‐affinity choline uptake mechanism, a common characteristic of cholinergic neurons. The Mabs themselves may possibly label some element of the high‐affinity transporter or a proximal membrane component. This implies that such a high‐affinity uptake mechanism is present in the subpopulation of NC cells at early times in development. If these cells in fact are destined to contribute to the avian CG, these characteristics are present in the subpopulation before the NC cells take on a neuronal morphology.
Establishment, characterization, and evolution of cultures enriched in type‐2 astrocytes
Authors: Aloisi F. , Agresti C. , Levi Giulio
Published: January 2005

The 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

To 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

The 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

Earlier 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

Proteins 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

The 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

Monoclonal 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

The 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

A 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

Fresh 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

Using 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

Sequence 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

Using 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.
Evidence that the axolemmal mitogen for cultured schwann cells is a positively charged, heparan sulfate proteoglycan‐bound, heparin‐displaceable molecule
Authors: Decoster M. A. , Devries G. H.
Published: January 2005

Treatment of axolemma with pH 9 buffer results in a pellet enriched two‐fold in the mitogen for cultured Schwann cells. Heparitinase treatment releases 8% of the mitogen into solution, while heparin selectively solubilizes the mitogen, resulting in an extract which has a specific mitogenic activity approximately 2.5 times greater than the mitogenicity of the starting axolemma membrane. These data support a model in which the axolemmal mitogen is a positively charged molecule associated with negatively charged sulfated proteoglycans.
Cytological and cytochemical (RNA) studies on rubral neurons after unilateral rubrospinal tractotomy: The impact of GM1 ganglioside administration
Authors: Barron K. D. , Banerjee M. , Dentinger M. P. , Scheibly M. E. , Mankes R.
Published: January 2005

The rubrospinal tract (RST) was cut unilaterally at C2–3 segment in 21 rats that were killed 3, 7, 10, 14, 28, 60, and 90 days later. Additionally, 14 rats, killed 14 or 28 days after lesioning, were treated postoperatively by daily intraperitoneal injections of GM1 ganglioside. Six unoperated, untreated rats served as controls. In untreated animals, axotomized neurons of the magnocellular division of the red nucleus (RN) exhibited cytoplasmic, nuclear, and nucleolar atrophy 7–10 days postoperatively. Atrophy progressed through the 90th postoperative day. Regression analyses disclosed a bimodal pattern to cytoplasmic and nucleolar atrophy, with an initial rapid phase changing to a slower but progressive mode from 14 days postoperatively. Nuclear atrophy proceeded in a unimodal manner. GM1 treatment did not affect these atrophic processes. Neuronal loss did not occur in the axotomized RN through the 60th postoperative day. Axotomized neurons of untreated rats showed significant and progressive reductions in mean somal (cytoplasmic) and nucleolar RNA from, respectively, the 7th and 14th postoperative day. GM1 partly prevented these RNA losses. Both in treated and untreated rats, spinal cord lesions contained many axonal sprouts 2 to 4 weeks after surgery, but newly generated axons did not traverse the rostro‐caudal extent of any lesion.
Consensus residues at the acetylcholine binding site of cholinergic proteins
Authors: Peterson G. L.
Published: January 2005

The nicotinic (nAcChR) and muscarinic (mAcCh) acetylcholine receptors and acetylcholinesterase (AcChEase) are structurally unrelated but share a common functional property: interaction with acetylcholine (AcCh). Alignment of the probable AcCh binding site regions of the nAcChR and mAcChR protein sequences revealed the presence of ten nearly identically spaced consensus residues, six of which contain potentially ligand‐interactive side chains. Important elements of the consensus residues also were found in one unique sequence region of the AcChEases. Alignments among the two receptors and AcChEase outside the apparent binding region were rare, and the consensus AcCh binding residues were largely substituted in the homologous proteins, which do not bind AcCh. The consensus residues include two possible anionic subsite Asp residues and a Ser that may hydrogen bond to the AcCh carbonyl in the receptors. These residues correspond to positions Asp‐166, Ser‐173, and Asp‐200 in the neuromuscular nAc‐ChR; Asp‐71, Ser‐78, and Asp‐105 in the M1 mAcChR; and Asp‐93 and Asp‐128 in Torpedo AcChEase. No corresponding consensus Ser is found in the AcChEase sequence; this is expected because of a downstream esterase active‐site Ser‐200 (Torpedo). A receptor‐conserved and disulfide‐linked Cys corresponding to neuromuscular nAcChR residue 193 and M1 mAcChR residue 97 may be important in energy transduction associated with agonist‐mediated events. The presence of additional binding‐site aromatic residues that may form a hydrophobic environment near the anionic subsite are aligned within, but not between, the three cholinergic protein groups. These observations target specific regions and residues within these proteins for structure‐function studies of the cholinergic binding domain.
Effects of free fatty acids and acyl‐coenzyme a on diacylglycerol kinase in rat brain
Authors: Kelleher J. A. , Sun G. Y.
Published: January 2005

Our earlier studies have indicated the presence of diacylglycerol kinase activity in rat brain cytosol as well as subcellular membrane fractions (Strosznajder et al.: Neurochemistry International 8(2):213–221, 1986). There is much evidence indicating the release of dincylglycerols due to stimulation of polyphosphoinositide hydrolysis by hormones and receptor agonists. In turn, diacylglycerols have been linked to a second messenger role for activation of protein kinase C. The present study tests the ability of free fatty acids and acyl‐coenzyme A (acyl‐CoA) to regulate diacylglycerol kinase activity. In a system containing brain cytosol and microsomes, addition of oleic acid (0.5 mM) resulted in large stimulation of diacylglycerol kinase activity as well as some translocation of the enzyme from cytosol to microsomes. On the other hand, oleoyl‐CoA (0.1 mM), but neither palmitoyl‐CoA nor arachidonoyl‐CoA, was effective in translocation of the diacylglycerol kinase. Unlike oleic acid, which preferred to associate with membranes, most of the oleoyl‐CoA remained in the cytosolic fraction. Since free fatty acids in brain are stringently controlled and are released during ischemic insult, a condition which also elicits the breakdown of polyphosphoinositide to diacylglycerols, results here suggest a plausible mechanism for regulation of diacylglycerol metabolism by free fatty acids and acyl‐CoA.
Different classes of cat spinal neurons display differential sensitivity to sodium pentobarbital
Authors: Paik K. S. , Nam S. C. , Chung J. M.
Published: January 2005

The effect of graded doses of systemically injected sodium pentobarbital on several classes of spinal neurons was studied using spinal cats. Classes of spinal neurons included unidentified dorsal horn cells, ascending tract dorsal horn cells, and motoneurons. Single unit activity of spinal neurons was evoked by electrically stimulating a peripheral nerve with an intensity strong enough to excite both A and C fibers. The A‐ and C‐fiber evoked activity was compared before and after intravenous injections of small incremental doses of sodium pentobarbital. The activity of different classes of spinal neurons showed different sensitivities to graded doses of systemically injected pentobarbital. The reflex activity of motoneurons elicited by stimulation of peripheral nerve was much more sensitive to pentobarbital than that of dorsal horn cells. In general, activity evoked by peripheral unmyelinated fibers was more susceptible to pentobarbital than was that evoked by myelinated fibers. However, intravenous injections of pentobarbital produced nondifferential suppression of dorsal horn cell activity evoked by noxious and innocuous mechanical stimuli applied to the peripheral receptive fields.
On the GABAA receptor: A molecular modeling approach
Authors: Aprison M. H. , Lipkowitz K. B.
Published: January 2005

Bicuculline methobromide, a complex alkaloid, antagonizes in some unknown manner the action of GABA, an important inhibitory transmitter in the CNS. To help understand the mechanism of this antagonism we have employed molecular modeling techniques to assess the similarity and difference between this antagonist and GABA plus four direct GABA agonists. Topological and electronic charge congruence between these five molecules was examined in great detail. It was found that each of the five molecules has three clearly defined atoms that serve as attachment points at the GABAA receptor site. It is hypothesized that an additional negatively charged atom on bicuculline serves as an additional point of attachment that blocks the nearby chloride ion channel. The model presented offers an explanation of why muscimol acts as a better agonist than GABA as well as rationalizing why (+)‐bicuculline acts as an antagonist but (−)‐bicuculline does not.
Ganglioside and lipid composition of bulk‐isolated rat and bovine oligodendroglia
Authors: Yu R. K. , Macala L. J. , Farooq M. , Sbaschnig‐Agler M. , Norton W. T. , Ledeen R. W.
Published: January 2005

We have examined the ganglioside composition of 30‐day and 60‐day postnatal rat oligodendroglia, adult bovine oligodendroglia, gray matter, white matter, and myelin and also the total lipid composition of the oligodendroglial preparations. The ganglioside patterns of rat and bovine oligodendroglia, as previously found for human oligodendroglia, were more complex than those of myelin. These data indicate that oligodendroglial perikarya can synthesize many brain type gangliosides, not all of which are incorporated into the compact myelin. Alternatively, the ganglioside composition of myelin may be altered in situ by the myelin‐associated neuraminidase. In these two species, as in human, GM4 appears specific to oligodendroglia and myelin, while GD3 and GM3 are enriched in oligodendroglia but not myelin. In bovine oligodendrocytes GD3 is the major ganglioside. The total lipid concentration, as well as the percentage of cholesterol, sphingomyelin, phosphatidylinositol, and phosphatidylserine, differ for 30‐ and 60‐day‐old rat oligodendroglia and may be developmentally correlated with changes in myelin composition during myelinogenesis. There are also marked differences in the lipid composition of bovine oligodendroglia compared to rat oligodendroglia, with the former having more galactolipid and less ethanolamine phosphoglycerides.
Implantation of rabbit embryo brain fragments into newborn mice: Integration and survival of xenogeneic astrocytes
Authors: Suard I. M. , Collins V. P. , Ignacio V. , Jacque C. M.
Published: January 2005

Brain fragments containing embryonic rabbit glia were implanted into the brains of newborn mice. The hosts developed an astroglial reaction around the transplants and along the needle tracks. Transplantderived astrocytes were identified in the operated brain by their expression of rabbit GFAP. During the first few days post‐implantation (PI) glial cells were exchanged between the transplant and the host. Less than 3 to 5 days PI, the transplant was extensively invaded by host astrocytes. Xenogeneic astroglial cells were first detected 10 days PI in the immediate proximity of the transplant. At 2 to 11 weeks, they could be detected either close to or at distance from the point of implantation. Most often, transplant‐derived astrocytes presented a morphology similar to that of neighboring host astrocytes. Xenogeneic glial cells were found to participate in various types of astroglial features: sub‐pial, pericapillary, fibrous, and protoplasmic. This morphological integration suggests that they are physiologically integrated, at least to a certain degree, in the host tissue.

In spite of their integration into the host, xenogeneic astrocytes disappear after 3 months without signs of an inflammatory reaction.
Modulation of pp60c‐src tyrosine kinase activity during secretion in stimulated bovine adrenal chromaffin cells
Authors: Oddie K. M. , Litz J. S. , Balserak J. C. , Payne D. M. , Creutz C. E. , Parson S. J.
Published: January 2005

High levels of the proto‐oncogene product, pp60c−src, have been found in developing and adult neural tissues as well as in certain fully mature cells of the hematopoietic lineage, e.g., platelets and myelomonocytes. Adrenal medullary chromaffin cells exhibit characteristics of both types of cells, i.e., they are derived from the neural crest and carry out exocytosis in response to specific stimuli. Earlier studies have shown that pp60c‐src localizes not only to the plasma membrane of chromaffin cells but also to the membranes of chromaffin granules, the secretory vesicles of these cells that store catecholamines and other secretory products. To investigate the possible involvement of pp60c‐src in exocytosis, cultured bovine chromaffin cells were analyzed for changes in c‐src tyrosine kinase activity in response to stimulation by several secretagogues. Results of in‐vitro immune complex kinase assays showed that pp60c‐src, derived from cells that had been stimulated for various lengths of time, exhibited decreased auto‐ and transphosphorylating activities as compared to pp60c‐src immunoprecipitated from control cells. The greatest reduction in activity was observed 10 min post‐stimulation, while normal levels were regained 2–6 hr after secretagogue treatment. Western immunoblot analysis of the immunoprecipitated pp60c‐src revealed that approximately 50% less c‐src protein was present in immune complexes prepared 10 min after stimulation as compared to those prepared from mock‐stimulated controls, resulting in a specific autophosphorylating activity that was 42–47% of control and little or no reduction in the transphosphorylating specific activity. In experiments in which the rate of secretion of [3H]‐norepinephrine from cells preloaded with this compound was compared to the rate of modulation of pp60c‐src activity, 50% of the maximal reduction in pp60c‐src activity occurred within 2–4 min while 50% maximal release of [3H]‐norephinephrine occurred within 1–3 min. Taken together, these results suggest that pp60c‐src may play some role (direct or indirect) in the exocytotic process.
Impact of the genetic background of transgenic mice upon the formation and timing of choroid plexus papillomas
Authors: Cho H. J. , Seiberg M. , Georgoff I. , Teresky A. K. , Marks J. R. , Levine A. J.
Published: January 2005

Transgenic mice harboring the SV40 large T antigen gene in a C57Bl/6J genetic background (SV11) first express this gene at 1–2 weeks of age, develop papillomas of the choroid plexus by 80–90 days, and die within 125 days after birth. Transgenic mice harboring the same transgene in a (SV11‐C57Bl/6J × NZW/lacJ) F1 genetic background express considerably lower levels of the transgene mRNA at comparable times after birth. As a consequence, tumor development and death are delayed. The NZW mice appear to contribute a dominant negative regulator for the expression of the SV40 large T antigen transgene, which in turn has a dramatic effect upon the time of appearance of tumors and the death of these transgenic animals.
Secretion of nerve growth factor in cultures of glial cells and neurons derived from different regions of the mouse brain
Authors: Houlgatte R. , Mallat M. , Brachet P. , Prochiantz A.
Published: January 2005

The regional ability of central neurons and glial cells to produce nerve growth factor (NGF) was studied in vitro. NGF secretion was compared in cultures of perinatal astrocytes or embryonic neurons that were derived from various mouse brain structures. No regional differences were detected among cultures of post‐natal day 2 glial cells of hippocampal, cortical, striatal, or mesencephalic origin. In all cases, levels of NGF released by the cells were very similar. They were closely correlated to the growth rate as shown by the fact that exponentially growing cells produced relatively more factor than did confluent cells, a finding in agreement with previous observations. Unlike growth‐phase cells, primary astrocytes immediately plated at high cell density did not secrete any assayable factor before the 7th day of culture. Levels of NGF found during the following days remained low. In contrast, striking differences were observed among cultures of embryonic neurons. NGF was found in relatively large amounts in cultures of embryonic day 17 or 19 striatal neurons, whereas media conditioned by neurons from the mesencephalon, cortex, or septum contained much less factor. Amounts of NGF assayed in cultures of hippocampal neurons varied with the time of sampling of this brain structure. Levels of factor were significantly higher in media conditioned by embryonic day 19 neurons than in media of embryonic day 17 neurons. However, amounts of NGF found in supernatants of hippocampal neurons remained smaller than those present in cultures of striatal nerve cells. Altogether, the results suggest that, in addition to astrocytes, central neurons may also synthesize and secrete NGF in vitro and that this phenomenum is dependent on both the origin and the developmental stage of the neuronal population.
Organization of oligodendroglial membrane sheets: II. Galactocerebroside: Antibody interactions signal changes in cytoskeleton and myelin basic protein
Authors: Dyer C. A. , Benjamins J. A.
Published: January 2005

Antibodies to galactocerebroside (GalC) cause patching of this surface glycolipid over internal domains of myelin basic protein (MBP), which are demarcated by a network of microtubules. The patching occurs whether or not second antibody is present, but the process is accelerated by the presence of second antibody. GalC patching results in disruption of microtubules in the lacy networks in oligodendroglial membrane sheets and in the eventual fusion of MBP domains, similar to the effects of colchicine (Dyer and Benjamins, 1989). Antibodies to GalC also disrupt F‐actin in the lacy networks. Since colchicine does not alter the distribution of F‐actin, anti‐GalC is causing F‐actin redistribution by a mechanism other than microtubule depolymerization. Extended exposure to anti‐GalC results in coalescence of surface GalC patches concomitant with fusion of internal MBP domains. When anti‐GalC is applied to induce GalC patching in cells previously treated with cytoskeletal inhibitors, patching is altered. After colchicine treatment, GalC surface staining is granular; i.e., patching is totally disorganized. Following cytochalasin B treatment, most membrane sheets display a few very large patches rather than the normal multiple, small patches. These GalC surface patterns are similar to the MBP distributions following the respective drug treatments (Dyer and Benjamins, 1989). Thus, the pattern of GalC distribution in the presence of antibody always reflects the organization of the underlying MBP domains; in turn, the organization of the MBP domains is determined by the lacy networks of microtubules in the oligodendroglial membrane sheets.