bluuebunny/crossref_metadata_2025 huggingface dataset:
Retinal morphology and visual pigment levels in 6‐and 12‐month‐old rhesus monkeys fed a taurine‐free human infant formula
Authors: Imaki H. , Jacobson S. G. , Kemp C. M. , Knighton R. W. , Neuringer M. , Sturman J.
Published: January 2005

Rhesus monkey infants were raised from birth until 6 or 12 months of age on a taurine‐free soy protein based human infant formula or on the same formula supplemented with taurine. An additional group received taurine‐free formula until 6 months. The densities of rod and cone visual pigments were measured by fundus reflectometry at 6 and 12 months, and retinal morphology was then examined by light and electron microscopy. The densities of rhodopsin, measured in the near periphery after a white bleach, and of cone pigment, measured in the macula after a red bleach, were significantly reduced in the taurine deprived monkeys at 6 months but not at 12 months. The retinas of 6‐month‐old taurine‐deprived infants showed degenerative morphological changes in photoreceptors, particularly in cones in the foveal region, which were somewhat less severe than those seen in a previous study at 3 months of age. The prevalence and degree of these abnormalities continued to decrease with age in taurine‐deprived animals, but changes persisted in some animals at 12 months. Recovery was more complete in monkeys reversed to the supplemented diet from 6 to 12 months. Thus, monkey infants are dependent on dietary taurine to maintain normal retinal structure until at least 6 months of age; the effects of taurine deprivation regress spontaneously but incompletely by 12 months. © 1993 Wiley‐Liss, Inc.
Morphine‐induced reciprocal alterations in Gαs and opioid peptide mRNA levels in discrete brain regions
Authors: Basheer R. , Tempel A.
Published: January 2005

The mechanisms involved in the development of morphine tolerance and dependence are still unknown. Recently much attention has been directed toward the changes in post receptor events. Opiate receptors, like other hormone and neurotransmitter receptors, have been shown to mediate their effects through guanine nucleotide binding proteins (G‐proteins). This, in turn, may cause alterations in intracellular events, one of which is transcription of specific genes. We investigated the changes in the levels of mRNA of proenkephalin (PPE) and prodynorphin (DYN) and the stimulatory G protein alpha subunit (Gαs) in adult morphine tolerant rats. Chronic morphine treatment induced reciprocal alterations in the levels of opioid peptide mRNA and Gαs in discrete brain regions. In striatum, PPE mRNA decreased by 49% (P < .01) and in hypothalamus. DYN mRNA showed a decrease of 21% (P < .01). In contrast, Gαs mRNA increased 20% (P < .01) in striatum and 97% (P < .01), in hypothalamus. In hippocampus the changes were reversed: PPE mRNA increased (55%, P <.05) and Gαs mRNA decreased (33%, P < .01). Frontal cortex exhibited a samll decrease in PPE (11.5%, P <.05) without any change on Gαs or DYN mRNA levels. These reciprocal alterations suggest an apposing mode of regulation of Gαs and PPE/DYN gene expression in morphine tolerant animals. © 1993 Wiley‐Liss, Inc.
Maintenance of glial plasticity with aging in C‐6 glial cells and normal astrocytes in culture: Responsiveness to opioid peptides
Authors: Kozlova M. , Kentroti S. , Vernadakis A.
Published: January 2005

In this study we used as glial cell models, early and late passage C‐6 glial cells, 2B clone, and advanced passages of glial cells derived from aged mouse cerebral hemispheres (MACH) to examine responsiveness to opioids. We have previously reported that early passage C‐6 glial cells, 2B clone, are bipotential and can be geared toward oligodendrocyte or astrocytic expression, whereas late passage C‐6 glial cells are astrocytic. In addition, MACH cultures have been previously characterized and consist of astrocytes type 1 and 2, some oligodendrocytes, and few glial precursors. In this study, early passage (17–20) and late passage (106–108) C‐6 glial cells or MACH cells of passages 16–19 were grown from plating time until harvesting, day 7 or 8, in DMEM + 10% FBS in the presence or absence of opioid peptides, Leu‐enkephalin (10−8 to 10−10 M) or its synthetic analog, dalargin (Tyr‐D‐Ala‐Gly‐Phe‐Leu‐Arg; 10−8 to 10−10 M). We examined for the activities of glutamine synthetase (GS) and cyclic nucleotide phosphohydrolase (CNP), enzyme markers for astrocytes and oligodendrocytes, respectively. We found that CNP activity was markedly increased in the early passage following opioid treatment, indicative of a shift to oligodendrocytic expression. In the late passage cells, already committed to astrocytic expression, opioid treatment enhanced GS activity suggesting that astrocytes respond to opioids. GS activity was markedly increased in MACH cultures grown in the presence of opioids with no changes in CNP. Thus, type 1 astrocytes, the predominant glial type in MACH cultures, responded to opioids. We conclude from these findings derived from two different glial models that regulation of astrocytes by microenvironmental signals appears to be maintained with aging. © 1993 Wiley‐Liss, Inc.
Cell‐type specific segregation of transcriptional expression of glial genes in the rat peripheral neurotumor RT4 cell lines
Authors: Hagiwara N. , Imada S. , Sueoka N.
Published: January 2005

Four types of cells, RT4‐AC (stem cell type), RT4‐B and RT4‐E (neuronal cell types), and RT4‐D (glial cell type) were previously isolated from an ethylnitrosourea (ENU) induced rat peripheral neurotumor RT4. In a phenomenon termed cell‐type conversion, RT4‐AC spontaneously and permanently gives rise to the three other cell types in culture. In the RT4 system the expression of glial fibrillary acidic protein (GFAP) and S100β protein genes segregates in a celltype specific manner. To further characterize the RT4 family, the expression of four myelin‐forming glial genes—P0 glycoprotein, suppressed cAMP inducible POU (SCIP), 2′, 3′‐cyclic nucleotide 3′‐phosphodiesterase (CNP), and myelin basic protein (MBP)—has been studied in the RT4 cell lines. In addition to these genes, the expression of the lowaffinity nerve growth factor (LNGF) receptor (expressed in immature Schwann cells) has been examined. We have found the following results. (1) The stem cell type RT4‐AC and the glial cell type RT4‐D express mRNA transcripts of P0, SCIP, and CNP (the larger form, 2.8 kb), and the amount of mRNA of these genes was increased by forskolin. (2) RT4‐AC and RT4‐D also express a low level of MBP mRNA upon forskolin treatment. (3) The neuronal cell types RT4‐B and RT4‐E do not express any of these myelinforming glial genes with or without forskolin treatment. (4) The LNGF receptor mRNA is expressed in RT4‐AC and RT4‐D and at a lower level in RT4‐B; its expression is stimulated by forskolin. © 1993 Wiley‐Liss, Inc.
Isolation and characterization of Bsk, a growth factor receptor‐like tyrosine kinase associated with the limbic system
Authors: Zhou R. , Copeland T. D. , Kromer L. F. , Schulz N. T.
Published: January 2005

Neuronal degeneration has been shown to be involved in various neurological disorders. Growth/trophic factors and their receptors are known to be important for the regeneration and survival of neurons. We report here the molecular cloning of a receptor‐like protein tyrosine kinase, bsk, (for brain specific kinase). Bsk is highly related to the eph/elk receptor like kinase family members. Northern blot analysis shows that is is expressed specifically in the brain, with no expression detected in adult heart, spleen, lung, liver, skeletal muscle, and kidney. In situ hybridization analysis of adult mouse brain sections indicates that bsk is expressed at high levels in the hippocampus, tenia tects, indusium griseum, and the piriform cortex, major components of the limbic system that are important for learning and memory. In addition, elevated levels of expression are found in other areas of the limbic system such as the amygdala, medial septum, and nucleus of the diagonal band, and in the olfactory bulb, which has close connections to the limbic system. The highest level of expression is found in the CA3 region of the hippocampus and the pyramidal cell layer of the piriform cortex. In 16.5 day mouse embryos, bsk is expressed predominantly in the primordial cortex of the telencephalon. An antibody against a C‐terminal peptide of bsk recognized a 105 kD protein in the 16.5 day embryonic head extract. Our analysis shows that bsk is a growth factor receptor‐like protein tyrosine kinase and that its greatest expression in the adult brain is associated with components of the limbic system. © 1994 Wiley‐Liss, Inc.
Glia‐derived nexin/protease nexin‐1 is expressed by a subset of neurons in the rat brain
Authors: Reinhard E. , Suidan H. S. , Pavlik A. , Monard D.
Published: January 2005

Glia‐derived nexin/protease nexin‐1 (GDN/Pn‐1) is a serine protease inhibitor that is secreted by glial cells and fibroblasts in culture. In the adult mammalian nervous system it has been shown to be expressed in the olfactory system and by some glial cells in response to neuronal injury. In situ hybridization and immunocytochemical studies were performed to identify the structures expressing GDN/PN‐1 in the developing and adult rat brain. In contrast to a transient widespreat expression during pre‐and postnatal development, some brain structures constitutively express GDN/PN‐1. These include the olfactory nerve layer of the olfactory bulb, basal forebrain, striatum, pyramidal neurons of layer V in the cortex, thalamic nuclei, pars compacta of the substantia nigra, inferior and superior colliculi, and deep cerebellar nuclei. All of these parts, excluding the olfactory nerve layer, are characterized by a high neuronal cell density. Neurons in these regions were immunoreactive for GDN/PN‐1. Furthermore GDN/PN‐1 expression in cell lines showed that the active protein was synthesized and secreted from B104 but not from NB2a neuroblastoma cells. Although GDN/PN‐1 has only been reported to be synthesized by glia, the results presented here demonstrate that in addition, a subset of neurons express this protease inhibitor. © Wiley‐Liss, Inc.
Involvement of protein kinase C in cAMP regulation of myelin basic protein gene expression
Authors: Anderson S. , Miskimins R.
Published: January 2005

Since synthesis of myelin components has been seen to be stimulated by cAMP in both oligodendrocytes and Schwann cells we have begun investigating the specific sequence(s) in the 5′ flanking region of the myelin basic protein (MBP) gene that are responsible for the induction of MBP transcription by cAMP. Using stably transfected cell lines containing various deletions of the MBP promoter directing the bacterial chloramphenicol acetyltransferase (CAT) gene we have identified a region of the MBP gene that is inhibitory to stimulation by increased cAMP levels. This inhibition can be overcome by pretreating the cells with 12‐O‐tetradecanoylphorbol 13‐acetate (TPA) for 48 hr. The effects on MBP gene expression modulated by TPA and cAMP involve altered DNA‐protein interactions in the 5′ end of the MBP promoter. The effect of TPA also appears to be mediated by down‐regulation of protein kinase C. © 1994 Wiley‐Liss, Inc.
Neuropeptide role of both peptide YY and neuropeptide Y in vertebrates suggested by abundant expression of their mRNAS in a cyclostome brain
Authors: Söderberg C. , Pieribone V. A. , Dahlstrand J. , Brodin L. , Larhammar D.
Published: January 2005

The evolution of the neuropeptide Y (NPY) family of peptides has been unclear despite sequence information from many vertebrates. We describe here two NPY‐related peptides deduced from cDNA clones of the river lamprey (Lampetra fluviatilis), a cyclostome providing one of the best models of a primitive vertebrate brain. One peptide corresponds to NPY as it has 83% identity to human NPY and its mRNA is expressed in the lateral brainstem, dorsal spinal cord and retina. The second lamprey peptide corresponds anatomically to peptide YY (PYY) as its mRNA is found in gut cells and in medial brainstem neurons. Its sequence is 60–70% identical to both PYY and NPY of mammals. These data suggest that the gene duplication leading to NPY and PYY had already occurred in the ancestral vertebrate 450 million years ago. The expression of the presumed PYY homolog in both gut and central nervous system indicates that PYY has served the dual role as a hormone and a neuropeptide from an early stage in vertebrate evolution. The similarities in the location of NPY‐ and PYY‐expressing cells between lamprey and mammals suggest that the functions of these peptides may have been conserved. © 1994 Wiley‐Liss, Inc.
Nerve growth factor regulates the subcellular localization of the nerve growth factor‐inducible protein PC4 in PC12 cells
Authors: Guardavaccaro D. , Montagnoli A. , Ciotti M. T. , Gatti A. , Lotti L. , Di Lazzaro C. , Torrisi M. R. , Tirone F.
Published: January 2005

The immediate early gene (IEG) PC4, which encodes a protein related to γ interferon, is activated at the onset of the neuronal differentiation induced by nerve growth factor (NGF) in PC12 cells. With an antibody raised to a bacterial β gal‐PC4 fusion protein, the PC4 protein is detected as an immunoreactive molecular species of 49 kDa, whose synthesis is rapidly induced by NGF in parallel with the induction of its mRNA. Immunofluorescence, electron microscopy and subfractionation studies indicate that the PC4 immunoreactivity is localized in the cytoplasm of PC12 cells, where it is increased transiently by NGF within 3 hr of treatment. In addition, the PC4 immunoreactivity presents an NGF‐dependent pattern of intracellular localization. In fact, within 3 hr after addition of NGF, PC4 is also significantly expressed on the inner face of the plasma membrane, to which it is physically associated. After longer NGF treatment, PC4 disappears from the plasma membrane and appears in the nucleus, with reduced cytoplasmic expression. Localization in the nucleus is reversed by removal of NGF and closely parallels changes in the state of differentiation of the cell. The existence within the PC4 protein of a consensus sequence for the addition of myristic acid and of a putative sequence for the nuclear localization suggests possible mechanisms for the NGF‐dependent redistribution. For an NGF‐inducible IEG product, such growth factor‐dependent localization of PC4 is a novel type of regulation in the pathways from the NGF receptor to the adjacent membrane proteins and to the nucleus. © 1994 Wiley‐Liss, Inc.
Involvement of glutamate receptors, lipases, and phospholipases in long‐term potentiation and neurodegeneration
Authors: Farooqui A. A. , Horrocks L. A.
Published: January 2005

Glutamate is the primary excitatory amino acid in the mammalian central nervous system. Normal excitation of glutamate receptors initiates the stimulation of phospholipases and lipases with the generation of second messengers that are necessary for normal cell function. The overstimulation of glutamate receptors can initiate a cascade of biochemical events including stimulation of membrane phospholipid turnover, excessive calcium entry, abnormal phosphorylation, and proteolysis. These events may be responsible for neuronal injury and degeneration found in Alzheimer disease, ischemia, spinal cord trauma, epilepsy, and Huntington disease.
L‐glutamate‐stimulated taurine release from rat cerebral cultured astrocytes
Authors: Koyama Y. , Ishibashi T. , Tanaka K. , Baba A.
Published: January 2005

We characterized L‐glutamate‐stimulated taurine release from cultured astrocytes prepared from rat cerebrum. L‐glutamate (0.5 mM) stimulated release of 3H‐labeled and endogenous taurine, where the rate of release reached maximum in 40 min. L‐glutamate increased astrocytic volume [3H‐O‐methyl‐D‐glucose (3H‐OMG) space] with a similar time course to 3H‐taurine release. Quisqualate, L‐aspartate, DL‐homocysteate, and L‐cysteate increased both astrocytic 3H‐OMG space and 3H‐taurine release from cultured astrocytes, while kainate (1 mM) stimulated 3H‐taurine release without affecting astrocytic volume. N‐methyl‐D‐aspartate had no effect on 3H‐taurine release and astrocytic volume. Treatment of astrocytes with dibutyryl cAMP reduced the effect of kainate on 3H‐taurine release. L‐glutamate‐stimulated 3H‐taurine release was attenuated by removal of extracellular Cl− and in hyperosmotic medium, which prevented L‐glutamate‐induced increase in 3H‐OMG space of cultured astrocytes. These results indicate that L‐glutamate stimulates taurine release from astrocytes through swelling‐triggered mechanisms and that kainate causes the release through volume‐independent mechanisms. © 1994 Wiley‐Liss, Inc.
Study of the phorbol ester effect on Alzheimer amyloid precursor processing: Sequence requirements and involvement of a Cholera toxin sensitive protein
Authors: Efthimiopoulos S. , Felsenstein K. M. , Sambamurti K. , Robakis N. K. , Refolo L. M.
Published: January 2005

Phorbol esters (PDBu) stimulate α‐secretase cleavage and secretion of the Alzheimer amyloid precursor protein (APP). To determine whether any cytoplasmic residues or sequence motifs mediate the PDBu effect on APP processing, this region of APP was altered by point mutations or deletions. To differentiate the mutated APP from the endogenous APP, the APP751 ectodomain between amino acids 1 and 647 was replaced by a human secreted alkaline phosphatase derivative (SEAP). The resultant fusion protein (SEAP‐APP751) was cleaved by α‐secretase at the same site as full‐length APP, and its secretion was stimulated by PDBu at a level similar to APP751. However, PDBu‐stimulated secretion of the SEAP‐APP751 fusion protein reached its maximum level after 30 min of treatment, while secretion of APP751 reached its maximum after 60 min, suggesting that the APP ectodomain affects the kinetics of APP secretion.

Mutation of the cytoplasmic serines to alanines had no effect on the PDBu‐stimulated secretion of the SEAP‐APP, indicating that protein kinase C (PKC) phosphorylation of the cytoplasmic domain of APP is not important for stimulation of APP secretion. Similarly, deletion of the cytoplasmic domain between amino acids 719 and 751 had no effect on the PDBu‐stimulated secretion. However, deletion of amino acids 707–751 resulted in a significant increase in the secretory cleavage of the SEAP‐APP707ΔC construct, suggesting that the sequence 707–719 is important for the regulated secretion of APP.

Cholera toxin, but not pertussis toxin, reduced the PDBu‐induced secretion of APP by more than two‐fold, suggesting that the PDBu response may be modulated by a cholera toxin sensitive heterotrimeric G‐protein. © 1994 Wiley‐Liss, Inc.
Preparation and affinity purification of a novel, biologically active, CNTF fusion protein
Authors: Rabinovsky E. D. , Browder D. P. , McManaman J. L.
Published: January 2005

A biologically active fusion protein comprising a short hydrophilic leader peptide fused to the N‐terminus of rat CNTF was generated using commercially available materials. The coding region for rat CNTF was sub‐cloned into the pFLAG‐1 vector and transfected into the JM 109 strain of E. coli. The transfected cells expressed high levels of the fusion protein (FLAG‐CNTF) following induction by isopropyl β‐D‐thiogalactoside (IPTG). FLAG‐CNTF is expressed as insoluble material that was resolubilized by extraction with guanidine hydrochloride and purified by immuno‐affinity chromatography. Analysis of the purified material by reverse phase HPLC and Western blot analysis indicated that FLAG‐CNTF was composed of two closely related species that are greater than 99% pure after affinity chromatography. The purified FLAG‐CNTF migrated as a 27 KD doublet on SDS polyacrylamide gel electrophoresis. Both bands of the doublet were shown to contain the FLAG peptide and CNTF by Western blot analysis, and amino acid sequence analysis demonstrated a single amino acid sequence corresponding to FLAG peptide and the N‐terminus of CNTF. The purified fusion protein was tested for biological activity using the IMR‐32 human neuroblastoma cell line. Treatment of IMR‐32 cells with FLAG‐CNTF increased the level of choline acetyltransferase (ChAT) in these cells 2–3‐fold over that of control cells in a dose dependent manner. A direct comparison of the effects of FLAG‐CNTF and recombinant human CNTF on IMR‐32 ChAT activity showed that both factors exhibited similar potencies. Our studies demonstrate the use of simple, commercially available, recombinant technology to efficiently generate large quantities of purified, biologically active CNTF. © 1994 Wiley‐Liss, Inc.
Basic fibroblast growth factor improves recovery after chemically induced breakdown of myelin‐like membranes in pure oligodendrocyte cultures
Authors: Fressinaud C. , Vallat J. M.
Published: January 2005

The putative role of growth factors in remyelination was investigated in pure oligodendrocyte (OL) secondary cultures derived from newborn rat brain. These cells form myelin‐like membranes and were used as a model system for toxic attack. A 24 hr treatment with 2.10–5 M lysophosphatidylcholine (LPC) induced a loss of 59% of the cells in these cultures, with a 64% reduction in [125I]‐iododeoxyuridine incroporation compared to untreated controls. An absence of processes and myelin‐like sheaths was observed in the remaining cells. Numerous intractoplasmic inclusions were observed on transmission electron microscopy. Immunocytochemical studies with A2B5 monoclonal antibody (mAb), which recognizes oligodendrocyte‐type 2 astrocyte (O‐2A) precursors, OL‐1 mAb (directed against cell surface sulfatides), and anti‐myelin basic protein (anti‐MBP) antibody showed that the entire OL lineage was affected at all stages of maturation. A 3 day treatment with 10 ng/ml basic fibroblast growth factor (bFGF) induced reconstruction of myelin‐like membranes, albeit less compacted than in untreated controls. The doubling in number of cells and the 46% increase in [125I]‐iododeoxyuridine incorporation was due essentially to proliferation of O‐2A progenitors. These results indicate that if bFGF release occurs during demyelination, it may participate in myelin repair mechanisms in the central nervous system. © 1994 Wiley‐Liss, Inc.
Secretion of nerve growth factor from septum stimulates neurite outgrowth and release of the amyloid protein precursor of Alzheimer's disease from hippocampal explants
Authors: Clarris H. J. , Nurcombe V. , Small D. H. , Beyreuther K. , Masters C. L.
Published: January 2005

Alzheimer's disease (AD) is characterized by the deposition of amyloid in the extracellular and intracellular compartments of the cerebral cortex. The extracellular amyloid consists of a protein (βA4) which is derived from a larger precursor, the amyloid protein precursor (APP). Several studies have implicated APP in the regulation of neurite outgrowth during development, although the precise function of APP remains unknown. To examine the role of APP in the regulation of neutrite outgrowth from hippocampal neurons, an explant culture system was developed. Explants of E18 mouse hippocampus were found to extend neurites when co‐cultured with explants of E18 mouse septum. This finding demonstrated that the septum can release a neurite outgrowth‐promoting factor (NOPF). As nerve growth factor (NGF) was also able to stimulate neurite outgrowth from the hippocampal explants, this suggested that the NOPF might be NGF. Immunoprecipitation of NGF from septal conditioned medium using a specific monoclonal antibody (27/21) completely blocked the neurite outgrowth‐promoting effect, supporting this conclusion. Concomitant with its ability to stimulate neurite outgrowth, NGF stimulated the release of APP from the hippocampal explants. As previous studies have suggested that the binding of APP to heparan sulfate proteoglycans (HSPGs) in the extracellular matrix might be an important step in the regulation of neurite outgrowth by NGF, we examined the effect of APP on neurite outgrowth from dissociated hippocampal cells cultured on various protein substrates. When cells were cultured on a substrate of APP and HSPG, neurite outgrowth was markedly stimulated. No stimulation of neurite outgrowth was seen when neurons were cultured on substrates of either APP or HSPG alone. The results suggest that secreted forms of APP may be involved in stimulating neurite outgrowth from hippocampal neurons and that interactions between APP and HSPG may be important for a neurite outgrowth‐promoting function. © 1994 Wiley‐Liss, Inc.
Reactive astrocytes are widespread in the cortical gray matter of amyotrophic lateral sclerosis
Authors: Nagy D. , Kato T. , Kushner P. D.
Published: January 2005

The distribution of reactive astrocytes was examined in the cortical gray matter of non‐motor and motor regions from cases of familial and sporadic amyotrophic lateral sclerosis (ALS) and compared to that of β‐amyloid deposits. By glial fibrillary acidic protein immunocytochemistry, patches of reactive astrocytes, characterized by multiple reactive astrocytes in a circular or patch‐like formation, occurred in 12 of 15 ALS cases examined. These patches of reactive astrocytes were not restricted to the motor cortex but were found in the gray matter in ALS in all examined brain regions, including frontal, temporal, inferior parietal, cingulate, occipital, and motor cortices, from both familial and sporadic ALS cases. Reactive astrocytes were also found in the subpial region and at the gray/white matter junction. Because patches of astrocytes can occur in association with senile plaques, β‐amyloid was localized. By immunostaining, β‐amyloid deposits were observed in five of the 15 ALS cases: three cases had only early plaques, two had both early and classic plaques. The number of ALS cases with both astrocyte patches and amyloid plaques was four of 15, but typically astrocyte patches in ALS occurred without any evidence of an association with β‐amyloid deposits. Therefore, the astrocyte patches in ALS are not the result of β‐amyloid deposition. The widespread occurrence of reactive astrocytes, as patches in the cortical gray matter and in the subpial region and at the gray/white matter junction, is evidence of a widespread pathology in ALS cortex in both familial and sporadic forms of the disease. © 1994 Wiley‐Liss, Inc.
Ubiquitin‐protein conjugates in different structures of the central nervous system of the rat
Authors: Adamo A. M. , Moreno M. Besio , Soto E. F. , Pasquini J. M.
Published: January 2005

The capacity to form ubiquitin (Ub)‐protein conjugates was investigated in the cytosol of different structures of the rat central nervous system (CNS) in order to confirm the presence of this extralysosomal, adenosine triphosphate (ATP)‐dependent, protein degradation system as well as its structural localization. Using 125I‐Ub, we found that in the presence of ATP, the cytosol obtained from whole brains was able to form high molecular weight Ub‐protein conjugates. These conjugates could be detected after sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) and radioautography. The formation of these conjugates was much higher in the cerebral cortex than in the brain stem, which is mainly constituted by white matter, being intermediate in the cytosol isolated from whole brain total homogenates. These results suggested to us that under normal conditions the capacity to form Ub‐protein conjugates was mainly located in structures containing neuronal cell bodies. Strong support for this contention was obtained when the cytosol isolated from rat optic nerves or from oligodendroglial cells isolated from whole brain was found to be totally unable to form Ub‐protein conjugates. The inability of certain CNS structures to form conjugates with Ub could be attributed, among other reasons, to the lack of enzymes catalyzing the various steps of the Ub degradation system, to the absence of short half‐life (target) proteins in those structures, or to the lack of activity of the enzymes catalyzing the reaction due to regulatory control mechanisms operating under normal conditions. © 1994 Wiley‐Liss, Inc.
VASE exon expression alters NCAM‐mediated cell‐cell interactions
Authors: Chen A. , Haines S. , Maxson K. , Akeson R. A.
Published: January 2005

The neural cell adhesion molecule (NCAM) is found on cells as several related polypeptides formed by alternative splicing of the single NCAM gene. The alternatively spliced 30‐bp VASE exon in the fourth immunoglobulin‐like domain is the structural variation nearest those portions of the polypeptide proposed to mediate cell‐cell adhesion. To test the ability of distinct forms of the NCAM molecules to mediate cell adhesion, L cells were transfected with expression vectors encoding rat 140 kD NCAM ± the VASE exon. L cell lines which expressed these polypeptides were isolated and tested for self‐aggregation in a low shear, rapid aggregation assay. Increased cellular aggregation of the transfectants was observed to be a function of the NCAM molecule expressed. These transfected cells showed segregation in a long term co‐aggregation assay: cells expressing NCAM — VASE formed aggregates which tended to exclude cells expressing NCAM + VASE and vice versa. These results provide direct evidence that this small difference in NCAM structure is sufficient to allow segregation of cells. © 1994 Wiley‐Liss, Inc.
Human neurocytoma cells in culture show characteristics of astroglial differentiation
Authors: Westphal M. , Stavrou D. , Nausch H. , Valdueza J. M. , Herrmann H.‐D.
Published: January 2005

Cultured human neurocytoma cells from two neurosurgical patients were analysed for their immunocytochemical staining patterns and growth characteristics. In both cases, the cells stained positive for glial acidic fibrillary protein (GFAP) within one day of tissue culture in medium, with and without fetal calf serum, whereas the histological tumor specimens were negative. Both cases contained cells concomitantly expressing GFAP and synaptophysin (SNP) in the primary cultures. Epidermal growth factor (EGF) was mitogenic for the cultured cells but not platelet derived growth factor alpha (PDGF AA) or nerve growth factor (NGF). It is concluded that the human neurocytomas may represent neoplasms of a pluripotent neuroglial cell which can provide an interesting model to study the determinants for human glial/neuronal differentiation in vitro. © 1994 Wiley‐Liss, Inc.
Long‐term effects of deprivation of cell support in the distal stump on peripheral nerve regeneration
Authors: Bajrović F. , Bresjanac M. , Sketelj J.
Published: January 2005

The distal stump of an injured peripheral nerve supports regenerating axons by offering a favourable growth substratum and several cell‐produced growth factors. Deprivation of cellular factors alone has been shown not to prevent fairly rapid axonal elongation after nerve injury if the growth substratum was preserved. The present study examined possible long‐term untoward effects of cell support deprivation during an early phase of nerve regeneration. Rat sciatic nerve was crushed and a 25 mm long distal nerve segment was made acellular by freezing‐thawing, while the integrity of the growth substratum for the regenerating axons was preserved. Toe‐spreading reflex and skin sensitivity to pinch in the foot were monitored to follow recovery of motor and sensory function, respectively. The number of myelinated axons was determined in the sciatic nerve proximally to the lesion site, and distally in the predominantly sensory sural nerve as well as in the mixed motor nerve to the soleus muscle. Except for a short delay in the onset of recovery, explainable by the reduced elongation rate of axons growing through the acellular nerve segment, we found no deleterious effect of cell support deprivation on sensory or motor function recovery after nerve crush. Most of regenerating sensory neurons did not critically depend on the distal stump cell support. However, a 15% and 25% loss of myelinated axons both proximally to the lesion and distally in the sensory sural nerve, respectively, indicated that a corresponding minor loss of injured sensory neurons occurred when they were deprived of such cell support even if provided with a favorable growth substratum for successful regeneration. In these animals, Dexamethasone application in a dose shown to reduce nerve growth factor (NGF) production in the proximal nerve segment did not detectably affect nerve regeneration. Under the same conditions of cell support deprivation, however, no axon loss could be demonstrated in the nerve to the soleus muscle, suggesting that the regenerating proprioceptive sensory neurons are less dependent on the distal stump cell support than the cutaneous ones. Copyright © 1994 Wiley‐Liss, Inc.
Fever and feeding in the rat: Actions of intrahypothalamic interleukin‐6 compared to macrophage inflammatory protein‐1β (MIP‐1β)
Authors: Myers R. D. , Lopez‐Valpuesta F. J. , Minñano F. J. , Wooten M. H. , Barwick V. S. , Wolpe S. D.
Published: January 2005

The chemokines, macrophage inflammatory protein‐1 (MIP‐1) and its subunit MIP‐1β, induce an intense fever in the rat when they are injected directly into the anterior hypothalamic, pre‐optic area (AH/POA), a region containing thermosensitive neurons. The purpose of this study was to compare the central action on body temperature (Tb) of MIP‐1β with that of interleukin‐6 (IL‐6), which also has been implicated in the cerebral mechanism underlying the pathogenesis of fever. Following the stereotaxic implantation in the AH/POA of guide cannulae for repeated micro‐injections, radio transmitters which monitor Tb continuously were inserted intraperitoneally in each of 15 male Sprague‐Dawley rats. Each micro‐injection was made in a site in the AH/POA in a volume of 1.0 μl of pyrogen‐free artificial CSF, recombinant murine MIP‐1β, or recombinant human IL‐6. MIP‐1β in a dose of 25 pg evoked an intense fever characterized by a short latency, a mean maximum rise in Tb of 2.4 ± 0.21°C reached by 3.7 ± 0.42 hr, and a duration exceeding 6.5 hr. Injected into homologous sites in the AH/POA, IL‐6 induced a dose dependent fever of similar latency and a mean maximal increase in Tb of 1.2 ± 0.25°C, 1.8 ± 0.15°C, and 2.1 ± 0.22°C and duration of 6.2 ± 1.28 hr, 6.7 ± 0.49 hr, and 6.8 ± 0.65 hr when given in doses of 25, 50, and 100 ng, respectively. These results show that MIP‐1β and the highest dose of IL‐6 induce a fever of comparable intensity, but MIP‐1β exerts its action in a much lower concentration. Thus, the de novo synthesis and subsequent action of the MIP‐1 family of cytokines on neurons of the AH/POA in response to a pyrogen challenge apparently play a functional role in the pathogenesis of fever. Further, the endogenous activity of IL‐6 in the hypothalamus which is enhanced in response to a lipopolysaccharide also may reflect its essential part in the acute phase response to a bacterial challenge. Copyright © 1994 Wiley‐Liss, Inc.
Expression of the putative pheromone and odorant transporter vomeromodulin mRNA and protein in nasal chemosensory mucosae
Authors: Krishna N. S. Rama , Getchell M. L. , Getchell T. V.
Published: January 2005

In nasal chemosensory systems, glandular proteins associated with the vomeronasal and olfactory epithelia perform specific perireceptor functions associated with sensory transduction. Vomeromodulin, a recently identified glycoprotein synthesized by the lateral nasal glands, is proposed to be a pheromone transporter (Khew‐Goodall et al., FASEB J 5:2976–2982, 1991). In our study, we have investigated its expression in vomeronasal, olfactory, and respiratory nasal mucosae of rats and humans using in situ hybridization and immunocytochemical techniques. In the rat, vomeromodulin mRNA and protein were localized abundantly in the glandular acini of the maxillary sinus component of the lateral nasal glands. In addition, the vomeronasal and posterior glands of the nasal septum also expressed vomeromodulin mRNA and protein. Vomeromodulin immunoreactivity was localized extracellularly in the mucus of the sensory and non‐sensory epithelia of the vomeronasal organ, and in the mucociliary complex of the olfactory, respiratory, and associated nasal epithelia. In human nasal mucosae, vomeromodulin immunoreactivity was localized in the mucociliary complex of the vomeronasal and respiratory epithelia. Comparison of the localization of vomeromodulin with that of odorantbinding protein, which is also synthesized in the lateral nasal glands of rats, revealed that odorant‐binding protein was expressed in a completely separate glandular region, namely the ventral component. In the septal glands, vomeromodulin was expressed in the posterior glands whereas odorant‐binding protein was localized in the anterior glands. Odorant‐binding protein immunoreactivity was not observed in the vomeronasal glands. In contrast, both proteins were localized in the mucus of vomeronasal, olfactory, and respiratory epithelia. Our results suggest that vomeromodulin, like odorant‐binding protein, functions as a chemosensory stimulus transporter associated with perireceptor processes in vomeronasal and olfactory transduction. Copyright © 1994 Wiley‐Liss, Inc.
Astroglia‐released factor shows similar effects as benzodiazepine inverse agonists
Authors: Rigo J.‐M. , Belachew S. , Lefebvre P. P. , Leprince P. , Malgrange B. , Rogister B. , Kettenmann H. , Moonen G.
Published: January 2005

Media conditioned by cultured neonatal cerebral cortex microexplants (CCM) or astrocytes (ACM) contain low molecular weight (<1,000 Da) substance(s) which inhibits the gamma aminobutyric acid (GABA)‐induced inward current recorded in cerebellar granule cells and hippocampal neurons in culture using the whole‐cell patch‐clamp technique. This effect is specific for CCm and ACM, as medium conditioned by PC12 cells (PC12CM) does not affect the GABA response of these cells. It is also specific for GABA‐induced currents because glutamate‐induced currents do not change either in amplitude or in shape in the presence of CCM or ACM. The inhibitory effect on the GABA response in cerebellar granule cells of both ACM and CCM could be suppressed by flumazenil, a specific benzodiazepine (BZD) antagnoist and could be mimicked by two BZD inverse agonists. These data thus demonstrate the presence of a BZD inverse agonist‐like activity in CCM and ACM. This effect of ACM on different neuronal cell types was heterogenous since no detectable effect could be observed on the GABA‐induced current in GABA‐responsive dorsal root ganglion (DRG) neurons, presumably reflecting a functional heterogeneity of the GABAA receptors present in these different neuronal subsets. By the release of such an endogenous BZD inverse agonist like activity, glia cells could possibly modulate GABAA receptor‐mediated responses. © 1994 Wiley‐Liss, Inc.
β/A4‐evoked degeneration of differentiated SH‐SY5Y human neuroblastoma cells
Authors: Lambert M. P. , Stevens G. , Sabo S. , Barber K. , Wang G. , Wade W. , Krafft G. , Snyder S. , Holzman T. F. , Klein W. L.
Published: January 2005

ß/A4 peptides are known to induce neurodegeneration in cultures of rat brain cells and rat neural cell lines (Yankner et al: Science 250:279–282, 1990; Behl et al: Biochem Biophys Res Commun 186:944–950, 1992). The current data show that these peptides induce similar neurodegeneration in SH‐SY5Y neuroblastoma cells, extending characterization of ß/A4 toxicity to a human nerve cell line. Human SH‐SY5Y cells respond to aggregated ß/A4 with changes in cell shape, membrane blebbing, antigenic modification, loss of attachment to the substrate, and cell death. ß/A4 peptides require aggregation for maximum toxic effects, as cellular degeneration is evoked by aggregated ß/A4 1‐42 and 4‐41 cysteine but not by monomeric ß/A4 1‐40. Aged (pre‐aggregated) ß/A4 1‐40 also evoked neurodegeneration. Antigenic changes comprise upregulation of Alzheimer's‐type tau epitopes, recognized by the PHF‐1 and Alz‐50 monoclonals. These particular changes in tau support the connectivity between this in vitro model and mechanisms leading to neurodegeneration in Alzheimer's disease. A significant feature of the SH‐SY5Y response is that cells must be differentiated before they become sensitive to the degeneration evoked by ß/A4. Signaling pathways leading to ß/A4‐evoked neurodegeneration thus are under experimental control, becoming complete only when proliferating cells withdraw from the cell cycle and develop a postmitotic phenotype. © 1994 Wiley‐Liss, Inc.
ω‐Contoxin binding sites and regulation of transmitter release in cerebellar granule neurons
Authors: Elster L. , Saederup E. , Schousboe A. , Squires R. F.
Published: January 2005

The protective action of Ca2+ and a series of other divalent cations on heat inactivation (48°C, 30 min) of [125I]ω‐conotoxin (CTX) binding sites was investigated in membranes prepared from rat forebrain. Moreover, the influence of GABA (500 ω) on this protection was studied. Binding of [125I]CTX as well as its inhibitory action on K+ (55 mM) stimulated, Ca2+‐dependent transmitter release were studied in rat cerebellar granule neurons cultured in the presence or absence of the GABAA receptor agonist THIP (4,5,6,7‐tetratydroisoxazolo[5,4‐c]pyridin‐3‐ol). In cells cultured in the presence of THIP (150 ω) it was investigated whether the ability of THIP to inhibit evoked transmitter release could be influenced by CTX. Ca2+ and other divalent cations could effectively protect against heat inactivation of [125I]CTX binding sites in rat forebrain membranes, but this protective action was not influenced by the presence of 500 ω GABA. The cultured cerebellar granule neurons exhibited specific binding sites for [125I] CTX, the number of which was independent of exposure of the cells to THIP during the culture period. Evoked transmitter release was inhibited by CTX with an IC50 value of 13 nM. In neurons cultured in the presence of 150 ω THIP, THIP could dose‐dependently inhibit evoked transmitter release, but this inhibitory action was not influenced by CTX (20 nM). The results show that cerebellar granule neurons exhibit functionally meaningful CTX binding sites. An association between such sites and GABA receptors is not apparent. © 1994 Wiley‐Liss, Inc.
Mitogenic effects of platelet‐derived growth factor, fibroblast growth factor, transforming growth factor‐β, and heparin‐binding serum factor for adult mouse Schwann cells
Authors: Watabe K. , Fukuda T. , Tanaka J. , Toyohara K. , Sakai O.
Published: January 2005

Mitogenic effects of fetal calf serum (FCS), platelet‐derived growth factor (PDGF), fibroblast growth factor (FGF), transforming growth factor‐β (TGF‐β), and forskolin to adult mouse Schwann cells were examined by bromodeoxyuridine (BrdU) incorporation and double immunofluorescence for S100 and BrdU. PDGF‐BB, basic FGF, and TGF‐β1 and β2 were all mitogenic for Schwann cells in media containing FCS. Forskolin suppressed the mitogenic activity of these factors. In serum‐free media, PDGF‐BB and bFGF were also mitogenic, but TGF‐β1 and β2 were not. Heparin‐binding fractions of FCS obtained by heparin‐Sepharose chromatography synergized with TGF‐β1 and β2 to produce a mitogenic response. Since PDGF‐BB, acidic FGF, and basic FGF were not detected in these fractions by immunoabsorption and immunoblot assays, the presence of unidentified heparin‐binding molecules in FCS bioactive for adult mouse Schwann cells is suggested. © 1994 Wiley‐Liss, Inc.
Induction of low‐affinity GABAA receptors by the GABA‐agonist THIP (4,5,6,7‐tetrahydroisoxazolo[5,4‐c]pyridin‐3‐ol) in cultured rat cerebellar granule cells is prevented by inhibition of polyamine sbiosynthesis
Authors: Abraham J. H. , Seiler N. , Schousboe A.
Published: January 2005

GABAA agonist‐induced formation of low‐affinity GABAA receptors in cultured cerebellar granule cells was studied in the presence or absence of α‐difluoromethylornithine (DFMO), a blocker of polyamine formation. High‐ and low‐affinity GABAA receptors were monitored by Scatchard analysis of [3H]GABA binding to membranes from cells cultured for either 4 or 10 days in the presence or absence of the GABA agonist 4,5,6,7‐tetrahydroisoxazolo[5,4‐c]pyridin‐3‐ol (THIP). Cultures grown for 4 days were exposed to THIP and DFMO for an additional period of 6 hr (acute exposure), whereas cultures grown for 10 days were exposed to the same agents during the entire culture period (chronic exposure). Regardless of the culture period or drug exposure protocol, control cells expressed only a high‐affinity (KD 7 nM) binding site for GABA, whereas the cultures treated with THIP for either 6 hr or 10 days exhibited an additional low‐affinity binding site (KD ∼ 500 nM). Chronic exposure to DFMO prevented the THIP induction of low‐affinity GABAA receptors, whereas acute exposure to DFMO had no effect on the ability of THIP to induce low‐affinity GABAA receptors. Measurements of the intracellular polyamine concentration demonstrated a slight decrease in the putrescine level in the granule cells exposed to DFMO or THIP + DFMO for 6 hr. In contrast, granule cells chronically (10 days) exposed to DFMO or THIP + DFMO were depleted of putrescine and spermidine. Hence, the ability of THIP to induce low‐affinity GABAA receptors was prevented by the simultaneous depletion of the cellular content of putrescine and spermidine, whereas inhibition of ornithine decarboxylase and of putrescine formation was not sufficient to prevent THIP‐induced receptor formation. © 1994 Wiley‐Liss, Inc.
Acute tolerance to the excitatory effects of opioids in the rat hippocampus
Authors: Angelopoulos E. , Koutsoukos E. , Maillis A. , Zioudrou C. , Stefanis C.
Published: January 2005

Prolonged iontophoretic administrations of δ‐ and μ‐selective opioid receptor agonists were conducted in the hippocampus of rats, in order to study the possible development of acute tolerance to the excitatory effects of the opioids. Acute tolerance (AT) to the excitatory effects of the 8‐selective opioid receptor agonist Tyr‐D‐Ser‐Gly‐Phe‐Leu‐Thr (DSLET) was observed when the drug was applied locally for 3–5 min in the CA1 hippocampal pyramidal neurons. The acute tolerance was expressed as a decrease in the commissurally evoked spike responsiveness during peptide's administration and led to a long‐lasting potentiation of the population spike (PS) upon its withdrawal. In all cases, where AT and spike potentiation were evident, the population excitatory postsynaptic potential (pEPSP) remained unaltered. Pharmacological studies of AT and long‐lasting spike potentiation showed the following: (1) the non‐selective opioid receptor antagonist, naloxone, while effective in blocking the excitatory effects of DSLET when applied prior and during the application of the latter, failed to exhibit and effect on the long‐lasting potentiating effect of the opioid; and (2) during the spike potentiation phase, administration of DSLET exhibited a depressant effect towards baseline values. This depressant effect of the opioid was evident 2–3 min from the beginning of the application and was completely antagonized by naloxone. The above results show that the development of acute tolerance to the excitatory effects of the DSLET led to long‐lasting spike potentiation, which manifests a withdrawal phenomenon. © 1995 Wiley‐Liss, Inc.
Insulin‐like growth factors protect against diabetic neuropathy: Effects on sensory nerve regeneration in rats
Authors: Ishii Douglas N. , Lupien S. B.
Published: January 2005

Neuropathy is an enigmatic and debilitating complication of diabetes. A consensus as to the pathogenesis of this disorder has yet to emerge. Recently, it has been found that the insulin‐like growth factors (IGFs) regulate peripheral nerve regeneration, and IGF content is reduced in various diabetic tissues. We tested herein the hypothesis that IGF administration can prevent or ameliorate the impairment of sensory nerve regeneration in streptozotocin diabetic rats. Miniosmotic pumps released small local doses of IGF‐I from a catheter routed near a site of sciatic nerve crush or larger systemic doses of IGF‐I or IGF‐II from a distant subcutaneous site. Whether administered locally or systemically, IGFs protected against the impairment of sensory nerve regeneration. Surprisingly, this protection was obtained despite unabated hyperglycemia. Therefore, the neuropathy involving sensory nerve regeneration in diabetes can be ameliorated or prevented by IGF treatment, independently of hyperglycemia. © 1995 Wiley‐Liss, Inc.