bluuebunny/crossref_metadata_2025_split huggingface dataset:
Spatial distribution of myelin basic protein mRNA and polypeptide in Quaking oligodendrocytes in culture
Authors: Barbarese E. 
Published: January 2005

AbstractIn the CNS, myelin is formed from the expansion of oligodendrocyte processes. In order to study myelin assembly in the hypomyelinating mutant mouse quaking (qk), cultures of Oligodendrocytes were established from affected and control animals. The cytoarchitecture of the Oligodendrocytes was analyzed by performing morphometric measurements after immunostaining with antitubulin. The results indicate that the gross morphology of the processes is similar in control and mutant cells. The localization of the message for the myelin structural component, myelin basic protein (MBP), was examined by in situ hybridization. In control Oligodendrocytes, 80% of MBP mRNA is found in the processes. In contrast, only 23% of MBP mRNA is localized to these structures in the mutant; the majority of MBP mRNA remains in the cell body. The mutant cells are capable of distributing mRNAs to the periphery as shown by the presence of tubulin mRNA in their processes. MBP poly‐peptide was visualized by immunofluorescence and found in the perikaryon, processes and membranous expansions of the control cells. In the mutant, it is largely confined to the perikaryon, reflecting the distribution of the mRNA. These results suggest that the localization of MBP polypeptide is achieved by restricting the distribution of its mRNA, and that MBP assembly into the myelin membrane occurs in the processes. This step appears to be blocked in qk oligodendrocytes in culture.
Expression of a novel gene product by transplants of genetically modified primary fibroblasts in the central nervous system
Authors: Doering L. C. , Chang P. L. 
Published: January 2005

AbstractPrimary fibroblasts initiated from skin biopsies of Wistar rats were transfected with a plasmid that encodes the human growth hormone and the neomycin resistance genes. Cell clones selected for G418 resistance and expressing high levels of human growth hormone were propagated in vitro and subsequently transplanted into the cerebral cortex of adult allogeneic rats. Grafts were examined by immunocytochemistry at weekly intervals up to 2 months. Fibroblasts in the transplants survived and expressed growth hormone clearly up to 4 weeks, but showed reduced expression at 6 to 8 weeks. Significant levels of human growth hormone were also detected by radioimmunoassay in the serum of the host rats up to 1 month. The experiments demonstrate that primary fibroblasts can be genetically modified to deliver a new gene product into the central nervous system, and the gene product can pass the blood‐brain barrier to enter the systemic circulation. This model illustrates the potential to introduce desired products into the brain through the genetic alteration of autologous primary fibroblasts.
Calcium‐activated neutral proteinase (CANP; calpain) activity in Schwann cells: Immunofluorescence localization and compartmentation of μ‐ and mCANP
Authors: Banik N. L. , DeVries G. H. , Neuberger T. , Russell T. , Chakrabarti A. K. , Hogan E. L. 
Published: January 2005

AbstractCalcium‐activated neutral proteinase (CANP) activity was determined in cytosolic and membranous subcellular fractions of transformed Schwann cells (tSc). The μM and mM Ca2+ ‐sensitive (μ‐and mCANP) forms of CANP were separated by DEAE and phenyl Sepharose column chromatography, the latter step enabling removal of the endogenous inhibitor calpastatin. The tSc contained more μCANP than the mM isoform. More than 75% of mCANP activity was membrane‐associated and 20% was cytosolic. In contrast, approximately 80% of μCANP was cytosolic and 15% was membranous. Triton X‐100 stimulated activity of the whole homogenate and of the membrane pellet but did not stimulate CANP activity in the cytosolic fraction. Immunohistochemical distribution of mM enzyme was studied in both fixed and permeabilized tSc with cytosolic (anti‐cyt‐mCANP) and myelin (anti‐my‐mCANP) antibodies. Live cells (non‐permeabilized) stained with anti‐my‐mCANP had a single filamentous ring circumscribing individual cells. Permeabilized cells treated with anti‐my‐mCANP had immu‐noreactive deposits throughout the intracellular space but sparing the perinuclear region. No immunohistochemical staining was detected when live cells were exposed to anti‐cyt‐mCANP whereas permeabilized cells had extensive intracellular staining with the most intense immunoreactivity in the perinuclear region. Our results indicate that both forms of CANP are present in tSc and that the activity of most of the μCANP is cytosolic while mCANP is particulate.
Distribution of protein kinase C isozymes in rat optic nerves
Authors: Komoly S. , Liu Y. , Webster H. deF. , Chan K.‐F. J. 
Published: January 2005

AbstractLight (LM) and electron (EM) microscopic immunocytochemical methods were used to study the distribution of protein kinase C (PKC) isozymes in adult rat optic nerves. In cryostat and vibratome sections examined by LM, type II (βrpar; isozyme was localized almost exclusively in the axons. In the EM, immunoreaction products were found to associate with microtubules and neurofilaments. The inner surface of axonal membranes were occasionally stained. Analysis of PKC isozyme composition of the optic nerves by using immunoblot techniques revealed that type II (β) isozyme accounted for approximately 80% of the total immunoreactivity. By contrast, type III (α) isozyme, which accounted for the remaining 20% of PKC, was found mainly in the astrocytes. Astrocytic processes next to blood vessels and between myelinated axons were stained. In the EM, immunoreaction products were found in the cytoplasm and along astroglial filaments. Segments of plasma membranes also were stained; but nuclei were unstained. Adult glial cells were not stained by an antibody to type II (β) isozyme except for the occurrence of a few punctate cytoplasmic densities in occasional astrocytes. Very faint or no immunostaining was observed in sections treated with a monoclonal antibody to type I (α) isozyme. Immunoblot analyses also did not reveal this subspecies. The absence of type I (α) isozyme in optic nerves is not due to a down‐regulation of the enzyme during development. In developing (5 and 11 day) rats, immunoreactivity of protein kinase C was very faint or absent. After 15 days, reaction products of both type III (α) and type II (β) isozymes were found throughout the nerve. These findings suggest that type II (β) isozyme may be involved in axonal transport whereas type III (α) isozyme may play a role in some astrocyte functions in mature optic nerves.
Identification of transcriptionally regulated genes after sciatic nerve injury
Authors: Leon M. De , Welcher A. A. , Suter U. , Shooter E. M. 
Published: January 2005

AbstractMammalian peripheral nerve fibres can regenerate after injury. In an attempt toward a better understanding of the underlying molecular events, we have isolated novel and known rat cDNA sequences, the expression of which are regulated during sciatic nerve regeneration. For this purpose, cDNA libraries were constructed from either the nerve segment distal to the crush site or the corresponding contralateral uninjured nerve of the same animals. These libraries were screened by differential hybridization and several transcriptionally repressed and induced sequences were isolated. Out of 2,000 cDNA clones screened from the distal library, 11 sequences were found to be induced in the distal nerve segment. This set of induced cDNAs included the rat homolog of vimentin, 28 S and 18 S ribosomal RNA species, and two novel sequences. Of 5,000 screened colonies of the contralateral library, 30 colonies contained sequences that were repressed in the distal segment after nerve crush. They were identified as myelin basic protein, myelin P0, α‐globin, cytochrome oxidase subunit 1, creatine kinase (muscle type, M) and collagen type I. In addition, five novel sequences were found that were dramatically repressed after sciatic nerve crush. Representative clones were tested by northern blot analysis to study their time course of transcriptional regulation during nerve regeneration. The observed patterns suggest that the regeneration phenomenon shows complex gene regulation in which the nonneuronal cells of the distal segment play an important role. Further characterization of the isolated regulated known and unknown sequences will increase our understanding of the molecular events associated with neuronal regeneration.
Glial growth is regulated by agonists selective for multiple opioid receptor types in vitro
Authors: Stiene‐Martin A. , Hauser K. F. 
Published: January 2005

AbstractTo determine whether one or more opioid receptor types might be preferentially involved in gliogenesis, primary mixed glial cultures derived from mouse ce‐rebra were continuously treated with varying concentrations of opioid agonists selective for mu (μ), delta DAGO ([D‐Ala2, MePhe4, Gly(ol) 5] enkephalin), (δ), i.e., DPDPE ([D‐PEN2, D‐PEN5]enkephalin), kappa (κ), i.e., U69,593, opioid receptor types. In addition, a group of cultures was treated with [Met5]‐enkephalin, an agonist for δ opioid receptors as well as putative zeta (ζ) opioid receptors. Opioid‐depen‐dent changes in growth were assessed by examining alterations in (1) the number of cells in mixed glial cultures at 3, .6, and 8 days in. vitro (DIV), (2) [3H]thymidine incorporation by glial fibrillary acidic protein (GFAP) immunoreactive, flat (type 1) as‐trocytes at 6 DIV, and (3) the area and form factor of GFAP‐immunoreactive, flat (type 1) astrocytes‐ DPDPE at 10−8 or 10−10 as M significant M enkephalin at 10−6, 10−8, 10−10 reduced the total number of glial cells in culture; but this effect was not observed with DAGO or U69, 593 (both at 10−6, 10−8 10−10) Equimolar or trations (i.e.), 10−6 M of [Met5]enkephalin U69,593, but not DPDPE or DAGO, suppressed the rate of [3H]thymidine incorporation by GFAP‐immunoreactive, flat (type 1) astrocytes. DAGO had no effect on growth, although in previous studies morphine was found to inhibit glial numbers and astro cyte DNA synthesis. [Met5]enkephalin (10−6 the only agonist to significantly influence astrocyte area. Collectively, these results indicate that δ (and perhaps μ) opioid receptor agonists reduce the total number of cells in mixed glial cultures; while [Met15]enkephalin‐responsive and perhaps κ‐respon‐sive) opioid receptors mediate DNA synthesis in astrocytes. This implies that δ opioid receptors, as well as [Met5]enkephalm, sensitive, non‐δ pioid receptors, mediate opioid‐dependent regulation of astrocyte and astrocyte progenitor growth. These data support the concept that opioid‐dependent changes in central nervous system growth are the result of endogenous opioid peptides acting through multiple opioid receptor types.
Some neural intermediate filaments contain both peripherin and the neurofilament proteins
Authors: Parysek L. M. , McReynolds M. A. , Goldman R. D. , Ley C. A. 
Published: January 2005

AbstractMammalian neurons and neuron‐like cultured cells express the neural intermediate filament (IF) proteins neurofilament (NF)‐L, NF‐M, NF‐H, and peripherin. To determine whether these proteins are found within the same 10‐nm filament, light and electron microscope immunocytochemistry using peripherin and NF‐specific antibodies was performed on PC12 cells, nervous tissue, and isolated neural filaments from the cauda equina. Double‐label immunofluorescence showed that peripherin and NF‐L, ‐M, and ‐H were found in identical filamentous patterns in interphase and mitotic PC12 cells. Furthermore, expression of mutant peripherin in PC12 cells disrupted not only the peripherin network but also NF‐containing filaments. Immunoelectron microscopy of PC12 cell cytoskeletons showed that peripherin and NF subunit proteins were found in the same filament. In situ, in the sciatic nerve, peripherin/NF‐L or peripherin/NF‐M/‐H double‐label immunofluorescence illustrates at least three types of nerve fibers: those containing NF only, those labeled predominantly for peripherin, and fibers in which peripherin and NF subunits were colocalized. Immunoelectron microscopy of filaments isolated from nerve roots comprising the sciatic nerve also showed the same three labeling patterns seen by light microscopy. Some neural IF appear to contain predominantly NF proteins or peripherin, but in others, both proteins are found within the same IF.
Maturation and aging of the axonal cytoskeleton: Biochemical analysis of transported tubulin
Authors: Tashiro T. , Komiya Y. 
Published: January 2005

AbstractChanges in solubility and axonal transport of tubulin during maturation and aging have been investigated using sciatic motor fibers of rats at 4, 7, 14, 30, and 80 weeks of age. One to six weeks after injection of L‐[35S]methionine into the spinal cord, labeled cytoskeletal proteins in consecutive segments of the sciatic nerve and the ventral roots were fractionated into soluble and insoluble forms by extraction in 1 % Triton at low temperature. In 4‐week‐old rats, the two forms of tubulin were transported coordinately in a single wave with the average rate of 2 mm/day. At 7 weeks of age, two components in tubulin transport were observed to develop, possibly reflecting the maturation of the axonal cytoskeleton. The slower main component (1.5 mm/day) contained most of the insoluble form together with the neurofilament proteins and the faster component (3 mm/day) was enriched in the soluble form. Though significantly different in composition, the two components correspond to slow component a (SCa) and slow component b (SCb) originally defined in the optic system. A progressive decrease in transport rates of both SCa and SCb was observed with rats at 14, 30, and 80 weeks of age. In addition, there was a large decrease in the proportion of insoluble tubulin during the course of transport in animals older than 30 weeks. This loss of the insoluble form seems to be accounted for partly by the proteolytic degradation of the severely retarded SCa proteins. Changes in axonal transport of tubulin may thus reflect age‐related changes in dynamics and turnover of the axonal cytoskeleton.
Identification of cytoskeletal, focal adhesion, and cell adhesion proteins in growth cone particles isolated from developing chick brain
Authors: Cypher C. , Letourneau P. C. 
Published: January 2005

AbstractGrowth cones are intimately involved in determining the direction and extent of neurite elongation during development They are able to monitor their environment and respond to it by undergoing directed motility. We have isolated a fraction enriched in growth cone particles from embryonic chick brain. Assayed by immunoblots, this fraction is enriched in GAP‐43, and contains the cytoskeletal proteins actin, myosin II, neurofilament protein, tubulin, kinesin, and dynamin. All of the major components of focal adhesions are also present: alpha‐actinin, vinculin, talin, and integrin. In addition to integrin, we also identify the cell adhesion molecules A‐CAM, L1, fibronectin, and laminin in these particles. This preparation of isolated growth cone particles may be a useful model system for studying growth cone adhesion and motility.
Compliance of hippocampal neurons to patterned substrate networks
Authors: Corey J. M. , Wheeler B. C. , Brewer G. J. 
Published: January 2005

AbstractNeuronal growth can be controlled in vitro by plating cells at low density and by differential adhesion between the cell and substrate. Primary cultures of rat hippocampal neurons were grown in serum‐free culture on polylysine‐coated glass coverslips patterned by selective laser ablation so as to leave grids of polylysine with varying linewidths (3, 5, and 10 μM), intersection distance (80, 120, and 160 μM), and nodal (intersection) diameter (5, 10, and 20 μM). Not only did somae strongly prefer the unablated polylysine areas, but they also migrated to foci where the local area of unablated polylysine was higher. These loci were the nodes, as opposed to the narrow connecting paths, and larger nodes, as compared with smaller nodes. Maximum migration to nodes of 88% occurred for a combination of 5‐μM pathwidth, 20‐μm node diameter, and 80‐μM pathlength. Daily observations indicated active migration to larger adhesive areas, which explains the differential compliance.
Epidermal growth factor exerts neuronotrophic effects on dopaminergic and GABAergic CNS neurons: Comparison with basic fibroblast growth factor
Authors: Ferrari G. , Toffano G. , Skaper S. D. 
Published: January 2005

AbstractBasic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) have been described to exert neuronotrophic effects on central nervous system neurons in culture. To study the selectivity of trophic actions of these growth factors, neurotransmitter‐identified populations of embryonic rat mesencephalon were used. At 20 days in vitro, EGF (3 ng/ml) promoted survival and neurite outgrowth from these neurons. The neuritogenic effect of bFGF (3 ng/ml) was, however, more robust. Quantitative analysis with the neurofilament monoclonal antibody RT97 and ELISA confirmed the differential response, bFGF being 2–2.5 times more effective at all concentrations tested (ED100:3–10 ng/ml for both EGF and bFGF). At 10 days in vitro, EGF displayed no trophic activity — even at 30 ng/ml. Treatment of mesencephalic cultures with EGF (3 ng/ml) for 20 days stimulated [3H]dopamine and [14C]GABA uptakes about 4‐fold. While bFGF (3 ng/ml) also stimulated GABA uptake some 4‐fold, dopamine uptake was increased almost 20‐fold. Thus, EGF is also capable of enhancing the transmitter traits of selected central neuronal populations; however, the actions of bFGF appear to preferentially address dopaminergic cells.
Effect of CNTF on low‐affinity NGF receptor expression by cultured neurons from different rat brain regions
Authors: Magal E. , Burnham P. , Varon S. 
Published: January 2005

AbstractOur previous work indicated that in E14 embryonic rat spinal cord cultures ciliary neuronotrophic factor (CNTF) exerted (1) a survival‐promoting effect on motor neurons and on a large population of unidentified neurons, and (2) a regulatory role on the expression of ChAT and low affinity NGF receptor (LNGFR) in a population of small/medium‐sized neurons. In the present study, we examined the effect of CNTF on the expression of LNGFR in cultures of different regions from the E18 embryonic rat brain, namely cortex, septum, striatum, mesencephalon, hippocampus, brainstem, and cerebellum. The number of LNGFR‐positive neurons (stained with the 192‐IgG monoclonal antibody) was determined in untreated cultures and in cultures treated for 6 days (0–6) with human recombinant CNTF. To distinguish between effects on survival and on LNGFR expression, experiments were performed in which CNTF was administered only for the last 48 h of the culture (from days 4–6). LNGFR positive neurons were found in the cultures of all the regions examined. In each one of them, CNTF increased the number of LNGFR‐positive neurons by three‐ to fourfold after 6 days of treatment. In the striatum, septum, mesencephalon, and cerebellum, the effect of CNTF was shown to be on the regulation of LNGFR expression and not on survival. In cultures from the cortex, hippocampus and brainstem, a survival‐promoting role of CNTF could be demonstrated. The effect of CNTF was dose dependent, with half‐maximal effects (ED50) achieved at 2–4.5 TU/ml for all the brain regions. Maximal effects were reached at 100–250 TU/ml. From these results, we conclude that (1) there exists a wide spectrum of CNTF‐responsive neurons in the central nervous system, and (2) CNTF plays an important and widespread role in regulating the expression of the LNGFR in neurons.
Peripheral nerve injury down‐regulates CNTF expression in adult rat sciatic nerves
Authors: Rabinovsky E. D. , Smith G. M. , Browder D. P. , Shine H. D. , McManaman J. L. 
Published: January 2005

AbstractCiliary neurotrophic factor (CNTF) is a 200‐amino acid protein expressed in high concentrations by peripheral nerves and is thought to be important for the survival and regeneration of injured motoneurons (Lin et al., J Biol Chem 265:8942–8947, 1990). To better understand CNTFs role in nerve injury we have characterized the effects of crush injury on the expression of CNTF in adult rat sciatic nerves using specific antibody and RNA probes. Following a crush injury, both the protein and mRNA levels undergo pronounced decreases distal to the crush. These changes in CNTF expression were qualitatively distinct from changes in the expression of the low‐af‐finity NGF receptor (p75NGFR), which increase following crush. Thus, the changes in CNTF levels do not reflect an overall down‐regulation of mRNA during degeneration, and are inconsistent with the proposed role of CNTF in neuronal injury, since its levels are decreasing at the same time as the requirement for neurotrophic support is increasing.
Further evidence for the dynamic formation of transmitter quanta at the neuromuscular junction
Authors: Vautrin J. , Kriebel M. E. , Holsapple J. 
Published: January 2005

AbstractFatt and Katz (Nature 166:597–598, 1950; J Physiol 117:109–128, 1952) attributed miniature endplate potentials (MEPPs) to the action of a standard quantity of transmitter, the quantum (Del Castillo and Katz, J Physiol 124:560–573, 1954). Quantal packets of transmitter were proposed to be preformed (Del Castillo and Katz, In CNRS Paris (Ed): “Microphysiologie comparée des éléments excitables” 67:245–258, 1957) and stored in large numbers in the motor nerve terminal. Statistical analyses of intervals between MEPPs and numbers of quanta composing small endplate potentials indicated that quantal release was a random process and that release sites functioned independently of each other. With the discovery of synaptic vesicles it was proposed that each contained one quantum of transmitter. The quantal‐vesicular hypothesis (Del Castillo and Katz, as cited above) fails, however, to explain amplitude distributions of MEPPs that are skewed and/or that show multiple peaks (Kriebel et al., Brain Res Review 15:167–178, 1990). The drop formation process (Shaw, “The Dripping Faucet as a Model Chaotic System,” Santa Cruz, CA: Aerial Press, Inc., 1984) was shown to generate amplitude classes of drops that were similar to classes of MEPPs which suggested that rapid changes in quantal size and ratios of skew‐ to bell‐MEPPs could be explained with a simple dynamic process which determines quantal size at the moment of release (Kriebel et al., as cited above, 1990). Further similarities between miniature endplate currents (MEPCs) and the formation of drops are reported here. We found that rapid changes in MEPC amplitudes and time courses, which accompany an increase in frequency, mimic changes in drop sizes that accompany increases in flow rate. MEPC intervals have a minimum and their distributions are comparable to those of drop intervals. During an increased rate of transmitter release, MEPP amplitudes and intervals were positively correlated. The results suggest that spontaneously released transmitter “packets” are formed at the moment of release and that transmitter supply to the process that forms packets is continuous. © 1992 Wiley‐Liss, Inc.