bluuebunny/crossref_metadata_2025 huggingface dataset:
Isolation and characterization of cDNA clones encoding epitopes shared with alzheimer neurofibrillary tangles
Authors: Dammerman M. , Goldstein M. , Yen S.‐H. C. , Shafit‐Zagardo B.
Published: January 2005

A pool of ten monoclonal antibodies of SDS‐insoluble epitopes of Alzheimer neurofibrillary tangles (NFT) was used to screen and adult brain cDNA expression library. Fourteen clones were isolated, two of which are described. The largest of the clones encodes 80 kD, or approximately 600 amino acids, of microtubule‐associated protein 2 (MAP 2). The MAP 2 region encoded by the cline shares at least two epitopes with human tau, another microtubule‐associated protein which cross‐reacts with NFT. In rat brain mRNA, the MAP 2 cDNA hybridizes to a single transcript of 9.5 kb. In human neuroblastoma mRNA, the MAP 2 cDNA hybridizes, at high stringency, to two transcripts of 9.5 kb and 6 kb. The 6‐kb transcript comigrates with the transcript for tau, as detected by a human tau cDNA. The properties of the MAP 2 cDNA suggest that, in humans, MAP 2 and tau have a common domain which may play a role in NFT formation. Another clone isolated with the anti‐NFT antibodies shares epitopes, but not nucleic acid homology, with the MAP 2 cDNA. This clone detects a single abundant transcript of 1 kb present in RNA from human neuroblastoma and from several non‐neuronal human cell lines. The properties of this cDNA suggest that it encodes a protein other than those previously reported to cross‐react with NFT.
Marcophate colony‐stimulating factor in nerve growth factor preparations
Authors: Erickson‐Miller C. L. , Abboud C. N. , Stach R. W. , Frantz C. N. , Brennan J. K.
Published: January 2005

Following a report that nerve growth factor preparations have granulocyte‐colony‐stimulating activity, we investigated the presences of colony‐stimulating factors in 7s mouse submaxillary nerve growth factor and its subunits. Macrophage colonies were formed in mouse bone marrow cultures after exposure to preparations of 7s nerve growth factor, the γ subunit, and, to a small extent, the α subunit: the β subunit, which is responsible for the nerve growth function, did not stimulate colony growth. Furthermore, the esteropeptidase activity of the γ subunit was not detected in preparations of macrophage colony‐stimulating factor purified from the giant cell tumor (GCT) cell line. Immunoprecipitation of radiolabeled γ subunit witha polyclonal antibody to L‐cell macrophage colony‐stimulating factor showed a protein band that could represent the γ subunit of nerve growth factor. Separation of the marcophage activity from the esteropeptidase activity of the γ subunit was accomplished on the basis of molecular size. Thus, macrophage colony‐stimulating factor was a contaminant of nerve growth factor produced by the mouse submaxillary gland and copurified with the γ subunit.
Occurrence of phospholipase A1–A2 and lysophosphatidylcholine acyltransferase activities in axolemma‐enriched fractions of brain stem, optic pathway, and cranio‐spinal nerves of the rabbit
Authors: Alberghina M. , Buonacera P. , Agodi A. , Stella A. M. Giuffrida
Published: January 2005

An axolemma‐enriched fraction was isolated and characterized from homogenates of brain stem, pooled optic nerve and tract, and sciatic and hypoglossal nerves of adult rabbits. In these fractions, the phospholipase A1 and A2, as well as the activity of acylCoA:2 ‐ acyl ‐ sn ‐ glycero ‐ 3 ‐ phosphorylcholine acetyl transferase, using 1‐acyl‐ and 2‐acyl‐GPC as acyl acceptors, were studied. The activity of the four enzymes was clearly detecable in the central nervous system (CNS) and peripheral nervous system (PNS) axolemmatic perparations, as well as in other subcellular fractions examined. The axolemma fracions, in which acetylcholinesterase displayed the highest activities, were particularlyenriched in the acylatin reaction enzymes. These latter showed specific activities about twofold higher compared with those of the homogenates and significant correlation with acetylcholinesterase. The noticeable presence of these enzymeactivities in both CNS and PNS axolemma suggests that a deacylation–reacylation system for phospholipids may be operative in this membrane.
Effects of arachidonic acid on respiratory activities in isolated brain mitochondria
Authors: Hillered L. , Chan P. H.
Published: January 2005

The present investigation was designed to examine the effects of free arachidonic acid (20:4),in concentrations relevant to cerebral ischemia, on brain mitochondrial respiratory activities and the reversibility of these effects. Incubation of brain mitochondria with 20:4causeda dose‐department increasein substratesupported (state 4) respiration (i.e., uncoupling) and a concomitant inhibition of substrate‐, phosphate‐, and ADP‐supported (state (3) or dinitrophenol‐supported state (3u) respiration. The temperature dependence of the 20;4 effects on mitochondrial respiration was also studied. It was found that the uncoupling and the respiratory inhibition were at least as pronounced at physiological temperatures as at room temperature. Arrhenius plots of the state 3 respiratory rates suggested that 20:4 did not cause a significant change in membrane fuidity. Addition of bovine serum albumin to the reaction medium following preincubation with 20:4 reversed the inhibition of state 3 respiration. The results suggesat (1) that 20:4mayinhibit mitochondrial ATP production during conditions of incomplete cerebral ischemia and 2) that 20:4 may limit the postischemic recovery of mitochondrial function.
Development of the amino acid pools in chick embryo brain, heart, and eye: Taurine, valine, glutamine, and phosphoethanolamine
Authors: van Gelder N. M. , Bélanger F.
Published: January 2005

The redistribution of valine, from the nonrenewable yolk supply into excitable tissues, was studied during the first 15 days of chick embryogenesis. Valine levels in the extraembryonic circulation (the vitelline plexus) peak between days 7–9 (E7–9) and then decline steeply. In their first phase of differentiation (E2–E4), all embryonic tissues contain more valine than the blood plexus. From E4 to E7, the heart and brain exhibit initially a rapid fall in valine, but from day 7 on the decrease becomes more gradual. The eye during the same period reaches an equilibrium with circulating valine; as these levels fall from E9 to E15, the eye retains the valine that accumulated.

Against this pattern of change, characteristic for an essential amino acid during embryogenesis, glutamine levels are at any time from two‐ to threefold higher than valine in all tissues. In the circulation, this ratio remains constant throughout the 15 days of embryonic development. Eye glutamine, higher on day 4, by E7 has entered into an equilibrium with glutamine in the plexus. A steady but two times higher glutamine level is maintained in the heart, although during the later stages of development it gradually tends to approach the plexus content. In sharp contrast, starting on E7 and accelerating on E9, a large increase of glutamine relative to valine or other essential amino acids is seen in developing brain tissue. This appears typical for most metabolic amino acids, suggesting that by days 9–10 the essential acid supply in the brain is being exhausted.

Phosphoethanolamine, present in very low concentrations in the nutritional supply, is actively extracted by the plexus and other embryonic tissues. By day 4, tissue levels are five times higher than in the circulation. From then on, apparently sequestered pools of phosphoethanolamine in the heart and eye gradually diminish as the embryo matures. The brain continues to accumulate the metabolite until day 9.

Taurine sequestration in the eye is saturated by day 4, and then appears to enter into an exchange equilibrium with the circulation. The heart steadily accumulates taurine during the entire embryonic period when levels are three to four times higher than in the plexus. Brain tissue is most active in taking up taurine, but the process has reached its limits by day 7. This limit is determined by the amount of taurine available in the yolk, since injections of taurine in the nutritional supply result on day 7 in a further increase in the brain. The heart, too, is able to sequester extra taurine but not before day 9 of embryogenesis. The eye, however, does not seem to develop a further capacity to sequester more taurine than is usually supplied by the circulation.
The effect of the experimental antitumor agent caracemide on brain choline acetyltransferase
Authors: Ho B. T. , Tansey L. W. , Feiffer R. , Newman R. A. , Farquhar D. , Field W. S. , Krakoff I. H.
Published: January 2005

Caracemide was found to inhibit choline acetyltransferase (CAT) from rat brain. A concentration of 0.5 mM caracemide inhibited the enzyme by 93%, whereas a degradation product from caracemide, N‐(methycarbamoyloxy)acetamide, produced only a 50% inhibition. Two other degration products, N(methyl‐carbamoyloxy)‐N′‐methylurea and N‐hydroxy‐N′‐methlurea, lacked any inhibitory activity. With bovine brain CAT, caracemide showed noncompetitive inhibitio with the substrate choline, Km337 μM, KiμM, Vmax2.83 nmol acetylcholine fromed/ mg/mg protein and mixed inhibition with the substrate acetyl‐CoA, Km21 μM, Ki146 μM, Vmax3.85 noml acetylcholine formed/min/mg porotein.
Neuroimmune intercommunication, central opioids, and the immune response to bacterial endotoxin
Authors: Dougherty P. M. , Dafny N.
Published: January 2005

Muramul dipeptide is the smallest biologically active fragment of the lipoplyasccharide (LPS) moiety of gram‐negative cell walls. The present report demonstrates that this product, associated with the immune response to bacterial infection, can modify CNS activity. Specifically, it is demonstrated that 6‐0‐stearoyl‐muramyl depeptide (MDP) can attenuate opiate withdrawal severity in a dose‐dependent fashion when injected into areas of the brain essential for this phenomenon. In additiion, MDP alters both baseline and postnarcotic electrophysiologic responses of four brain areas essential for various opioid activities. Similar findings have been reproted for interferon‐alpha (IFN‐α), a peptide associated with the immune response to virus. Yet, even though MDP and IFN are shown to exert similar effects on opioid activity, there are also some very distinct differences in the actions of both of these immune response products. These observations suggest that central opioid systems may provide targets for the perception as well as the differentiation of afferent immunologic sensory input to the brain.
The influence of laminin on the initial differentiation of cultured neural tube neurons
Authors: Heaton M. B. , Swanson D. J.
Published: January 2005

Portions of the metencephalic neural tube containing the trigeminal (V) motor nucleus from 40‐hr chick embryos were excised and held freely floating in culture medium for 36–40 hr, so that neuronal generation within motor V could be completed, but precluding neuronal differentiation. The explants were then dissocited and plated either on (1) glass coverslips that had been coated with the extracellular matrix (ECM) glycoprotein, laminin, and subsequently irradiated to produce a grid pattern; or (2) coverslips, one‐half of which had been coated with laminin, and the other one‐half with collagen, another component of the ECM. The purpose of these studies was to assess possible laminin influences on neuronal adhesion and nerve fiber expression and extension during these periods of initial neuronal differentiation. The early neural tube neurons selectively adhered to the established laminin grid pattern; neuronal survival, elaboration of neurites, and extent of neurites were significantly enhanced on the laminin side of the laminin/collagen preparations. These latter effects were specifically blocked by the application of anti‐laminin. In demonstrating these influences during stages of initial neuronal differentiation, the results support the hypothesis that laminin may play a role in normal neurogenesis, presumably by providing an adhesive surface for outgrowing growth cones.
Regulation of the number of α‐bungarotoxin binding sites in cultured chick myotubes by a 1,4 dihydropyridine calcium channel antagonist
Authors: Smilowitz H. , Smart E. , Bowik C. , Chang R.‐J.
Published: January 2005

Calcium has been suggested as the second messenger link between skeletal muscle activity and AchR gene expression and synthesis. We have compared the concentrations of the Ca2+ channel antagonists D600 and nisoldipine needed both to block Ca2+ uptake into cultured myotubes and to increase AchR expression. The good correspondence between these two measurements and the use of the highly specific Ca2+ channel antagonist nisoldipine strengthens the hypothesis that antagonist nisoldipine strengthens the hypothesis that AchR expression is regulated by levels of intracellular Ca2+.
Heterosis for myelin content is limited to the central nervous system
Authors: Miskimins R. , Yu R. K.
Published: January 2005

We have recently described a murine model for studying aspects of myelination (Seyfried and Yu, 1980; Ebato et al., 1983; Miskimins et al., 1986). This mouse shows hypermyelination during the period of most active synthesis of myelin, 9 to 21 days post‐natal. The myelin parameters showing an increase were all measured in the central nervous system. We investigated here whether this effect extends into the peripheral nervous system. Our results indicate that the hypermyelination is limited to the central nervous system.
Effects of insulin and‐like growth factor (IGF‐I)on oligodendrocyte‐enriched glial culture
Authors: van der Pal R. H. M , Koper J. W. , van Golde L. M. G. , Lopes‐Cardozo M.
Published: January 2005

The brains from 1‐week‐old rat pups were used to prepare cultures of glial cells. After 24 hr in culture the cells were changed to a chemically defined serum‐free medium (CDM). We have used antibodies against gangliosides (monoclonals A2B5 and LB1) and against galactocerebrosides to monitor the influence of insulin on the development of oligodenrocytes from precursor cells. In these oligodendrocyte‐enriched cultures we investigated the influence of insulin and IGF‐I on the activity of the marker enzymes 2′,3′‐cyclic‐nucleotide 3′‐phosphodiesterase (CNPase) and glycerol‐3‐phosphate dehydrogenase (G3PDH), and on lipid metabolism.

Incorporation of [35S] sulfate into sulfolipids was stimulated by insulin (optimal concentration 100 nM). A sharp peak in sulfolipid synthesis was seen at day 5–6in culture.

Insulin stimulated the expression of CNPase and G3PDH in culture.

The stimulating effect of insulin on sulfolipid synthesis, CNPase, and G3PDH activity was mimcked by IGF‐I (13 nM).

The incorporation of [35S] into sulfolipids and [2‐3H] acetate into fatty acids and cholesterol was reversibly reduced by temporary omission of insulin from the medium.

These results indicate that insulin and IGF‐I stimulate the development of oligodendroctes in our culture system and that insulin has a general anabolic effect on the lipid metabolism of the cells.
Rapid shrinkage of rat striatal extracellular space after local kainate application and ischemia as recorded by impedance
Authors: Korf J. , Postema F.
Published: January 2005

Early changes in tissue extracellur space following exposure to the excitotoxin kainate in the striatum were compared to those following cardiac arrest of rats anesthetized by chloral hydrate. Tissue extracellular space was monitored by impedance measurements. The possible role of voltage‐sensitive Na channels and energy metabolism was studied by local and systemic application of tetrodotoxine (TTX) and glucose, respectively.

After both kainate intoxication and cardiac arrest the extracellular space (normally about 20%) became less than one‐half within 15 min. TTX caused a delay in the effect of cardiac arrest, and a slight attenuation of that of kainate. Glucose was inffective in both preparation. Parallel to a decrease in the extracellular space whole tissue Na/K ratio increased.

These experiment show that excitotoxins and cardiac arrest cause similar (and not additive) change in the extracellular space and that these change are not mediated by na channel. In cardiac arrest oneset of the extracellular space alteration is triggered by Na+ influx, thus presumably by neurotransmitter release. It is suggested that most (if not at all) currently described protective measures against ischemic, hypoxic, or hypoglycemic brain damage are based on a prolongation of the time of onset leading to cell depolarization, rather than suppressing damaging processes during depolarization.
Activators of protein kinase C enhance cyclic AMP accumulation in cerebral cortical and diencephalic neurons in primary culture
Authors: Tapia‐Arancibia L. , Veriac S. , Pares‐Herbute N. , Astier H.
Published: January 2005

The effects of the active phorbol ester 12‐myristate, 13‐acetate (PMA), the inactive ester 4 α‐phorbol 12,13‐didecanoate (4 α‐PDD), and the synthetic diacylglycerol 1‐oleoyl‐2‐acetyl‐glycerol (OAG) on cyclic AMP production were examined in rat cerebral cortical and diencephalic cells. With the aid of a prelabeling technique for measuring cyclic AMP accumulation in the cells, it was found that neither PMA nor OAG significantly increased cyclic AMP formation in either type of cell. In contrast, PMA enhanced the cyclic AMP response to vasoactive intestinal peptide (VIP) and forskolin in cerebral cortical and diencephalic cells, whereas 4 α‐PDD was inactive. A 15‐min preincubation was used to obtain maximal enhancement. The concentration dependence of PMA on VIP‐stimulated cyclic AMP accumulation was determined in cortical cells (EC50 = 6.2 × 10−8M).

OAG was also able to potentiate VIP‐induced cyclic AMP formation in cortical and diencephalic cells. However, its potentiating effect was weaker than that observed with PMA treatment. The data show, at an early stage of development (primary cultures, 8–10 days), a modulation of VIP‐ or forskolin‐cyclic AMP response by the activators of protein kinase C, i.e., PMA and OAG, in two different structures of the central nervous system: the cerebral cortex and the diencephalon. To our knowledge, this is the first demonstration of such a potentiation within the diencephalon.
CNS neuronal cell line‐derived factors regulate gliogenesis in neonatal rat brain cultures
Authors: Bottenstein Jane E. , Hunter S. F. , Seidel M.
Published: January 2005

Recent studies suggest that heterotypic cell‐cell interactions influence gliogenesis in the developing rat central nervous system. CNS neuron‐derived factors have been hypothesized to exist, and several have been identified and partially characterized which affect the number of oligodendrocytes in vitro. In order to study further the role of neurons in gliogenesis, we have used serumfree culture conditions, the B104 CNS neuronal cell line as a source of soluble factors, and dissociated neonatal rat brain cells as a source of glial cells. We have analyzed the response of the glial cells to serum‐free B104 conditioned medium using morphological, immunocytochemical, autoradiographic, and enzymatic methods. Dose‐dependent increases in the number of morphologically identified oligodendrocytes occur in response to this conditioned medium. Galactocerebroside (GalC) is a specific marker for oligodendrocytes, and the A2B5 antigen marks bipotential glial progenitor cells and their progeny: immature oligodendrocytes and type 2 astrocytes. In the presence of conditioned medium, the number of cells expressing GalC and/or A2B5 antigen increases over time when measured at 4, 8, and 12 days in vitro. A significantly weaker effect is seen if serum is also present. Since the vast majority of A2B5‐positive cells in conditioned medium treated cultures lack glial fibrillary acidic protein (GFA), indicative of type 2 astrocytes, they represent glial progenitors and immature oligodendrocytes. Double immunostaining combined with autoradiography suggests that the latter cell types are the target cells for the oligodendrocyte‐promoting activity. In addition, the conditioned medium markedly increases 2′,3′ cyclic nucleotide 3′‐phosphodiesterase (an oligodendrocyte marker) and to a lesser extent enhances glutamine synthetase activity (an astrocyte marker). Type 1 astrocytes are also more morphologically differentiated in this condition, and their percentage is decreased simulataneously. Conditioned medium from other donor neural cells either has no activity or is much less effective than B104 conditioned medium. The active factors are soluble, sensitive to both trypsin and 100°C treatment for 20 min, and appear to be 30–100 kilodaltons by stirred cell ultrafiltration. In summary, we have identified a potent source of growith‐stimulating factors that produce increased numbers of glial progenitor cells and oligodendrocytes; the same conditioned medium also appears to inhibit type 1 astrocyte proliferation.
Molecular modelling of the immunoglobulin‐like domains of the neural cell adhesion molecule (NCAM): Implications for the positioning of functionally important sugar side chains
Authors: Santoni M.‐J. , Goridis C. , Fontecilla‐Camps J. C.
Published: January 2005

The neural cell adhesion molecule (NCAM) is thought to mediate cell–cell adhesion by a homophilic mechanism involving binding sites located in the N‐terminal region of the protein. This region of the molecule consists of five domains that are homologous to each other and share conserved residues with immunoglobulin domains. We report here secondary structure predictions for the five NCAM domains and three‐dimensional models for two of them. The results are entirely consistent with an immunoglobulin‐like folding of the NCAM domains into seven strands forming two b̃‐sheets. NCAM–NCAM binding may thus be analogous to the pairwise associations of immunoglobulin constant domains, which are involved in dimer formation. Insertions and deletions are located mostly in b̃‐turn regions. Two α‐helical regions in the third and fourth domain are predicted with high probability. NCAM bears two kinds of functionally important sugar side chains, sialic acid polymers in the fifth domain, which modulate NCAM binding, and the L2 moiety, which is involved in cell adhesion and can be assigned to the third domain. Three‐dimensional modelling of the corresponding domains indicates that two of the three sites for N‐linked glycosylation in the fifth and the single site in the third domain are located on he face of the domain, which in immunoglobulin contant regions engages in intermolecular interactions.
Expression of monoclonal antibody Q113 immunoreactivity in the rat cerebral cortex: Unique differential sublayering of layer I. Staining of radial glia
Authors: Plioplys A. V. , Hawkes R.
Published: January 2005

Monoclonal antibody mabQ113 recognizes a 120‐kilodalton polypeptide which, in the cerebellar cortex, is confined exclusively to a subset of Purkinje cells which are organized in parasagittal bands (Hawkes et al.: Brain Research 333:359–365, 1985). In all other areas of the adult rat brain examined the localization of the mabQ113 epitope was marked by regional neuronal and glial coexpression (Plioplys and Hawkes: Brain Research 375:1–12, 1986).

Similar neuronal‐glial co‐expression was characteristic of the adult rat cerebral cortex. Intriguingly, mabQ113 revealed a unique differential sublamination of layer I. In the neocortex, layer I was split into two sublayers, with the more superficial sublayer weakly stained and the deeper sublayer stained more intensely, whereas in the pyriform cortex, layer I was split into three. These sublaminations do not correspond to previously described subdivisions of layer I.

In the developing cortex, the mabQ113 epitope is found in radial glial fibers. Stained radial fibers are first seen beginning at E17, reach a maximum at P4 and finally disappear between P12 and P14. The laminar distribution of mabQ113‐immunoreactivity emerges earlier in the pyriform cortex than the neocortex: the sublamination of layer I is seen at P4 in the pyriform cortex but not until P8 in the neocortex.

The significance of these observations is discussed.
Macrophage‐like cells from explant cultures of rat sciatic nerve produce apolipoprotein E
Authors: Goodrum J. F. , Novicki D. L.
Published: January 2005

Apolipoprotein E is synthesized and secreted by degenerating peripheral nerve, but the role of resident endoneurial cells in this process is not clear. To exclude the involvement of nonresident cells, we examined the cellular source of endoneurial apolipoprotein E in explant cultures of rat sciatic nerve. The cellular outgrowth from these explant cultures released apolipoprotein E into the culture medium. The cellular outgrowth contained fibroblasts, Schwann cells, and a population of cells with many phenotypic characteristics of macrophages, including the production of apolipoprotein E. No other cell type in the cultures appeared to contribute to this production. These data suggest that apolipoprotein E is produced by resident endoneurial cells in explant cultures and that these cells are macrophages.
Nerve growth factor binding in aged rat central nervous system: Effect of acetyl‐l‐carnitine
Authors: Angelucci L. , Ramacci M. T. , Taglialatela G. , Hulsebosch C. , Morgan B. , Werrbach‐Perez K. , Perez‐Polo J. R.
Published: January 2005

The nerve growth factor protein (NGF) has been demonstrated to affect neuronal development and maintenance of the differentiated state in certain neurons of the peripheral and central nervous system (CNS) of mammals. In the CNS, NGF has sparing effects on cholinergic neurons of the rodent basal forebrain (BF) following lesions where it selectively induces choline acetyltransferase (ChAT). NGF also induces ChAT in the areas to which BF provides afferents. In aged rats, there is a reduction in the NGF‐binding capacity of sympathetic ganglia. Here, we wish to report that there is a decrrease in the NGF‐binding capacity of the hippocampus and basal forebrain of aged (26‐month‐old) rats as compared to 4‐month‐old controls but no change in NGF binding in cerebellum. In all instances, equilibrium binding dissociation constants did not differ significantly. Treatment of rats with acetyl‐L‐carnitine, reported to improve cognitive performance of aged rats, ameliorates these age‐related deficits.
Axotomy of the rat facial nerve leads to increased CR3 complement receptor expression by activated microglial cells
Authors: Graeber M. B. , Streit W. J. , Kreutzberg G. W.
Published: January 2005

Axotomy of the rat facial nerve leads to mitotic divisions of microglial cells without developing into phagocytes. In order to study the functional characteristics of those activated, i.e., proliferating but nonphagocytic, microglia we investigated the expression of monocyte/macrophage antigens by these cells. Our results show that activated microglia lack monocyte/macrophage antigens recognized by the monoclonal antibodies Ox‐41, ED1, ED2, and Ki‐M2R but express high levels of CR3 complement receptors in situ.
Organization and expression of the human myelin basic protein gene
Authors: Kamholz J. , Toffenetti J. , Lazzarini R. A.
Published: January 2005

The human brain contains four isoforms of myelin basic protein (MBP), previously identified by cDNA cloning. We have now isolated and characterized genomic clones encoding the human MBP gene. The gene is 45 kb in extent and consists of seven exons. Alternative splicing of the primary MBP transcript can account for all four human MBP isoforms. The intron‐exon boundaries of the gene have also been determined, and all conform to the known consensus splice sequences. These sequences, however, do not explain the alternative splicing pattern found in human brain.

Transcription of the human MBP gene begins at a single site within the MBP promoter, and all four MBP isoforms are transcribed from this same site. The promotor region does not contain any known sequence elements, but does have a 12‐bp sequence also found in the JC virus 98‐bp tandem repeat. A relative gradient of MBP transcription is found from caudal to rostral within the developing human brain, which parallels the known sequence of myelination found in these areas. RNase protection of brain RNA demonstrates more of the 21.5‐kD and 20.5‐kD MBP mRNAs in neonatal brain than in the adult frontal cortex, which suggests that alternative splicing of the primary MBP transcript is also regulated temporally during myelin development. These data show that regulation of myelination is complex, involving regional cellular interactions and trans activation of transcription, as well as modulation of alternative splicing. Comparison of the human and mouse data also suggests that alternative splicing plays an important role in myelin biogenesis.
Basic fibroblast growth factor and epidermal growth factor exert differential trophic effects on CNS neurons
Authors: Morrison R. S. , Keating R. F. , Moskal J. R.
Published: January 2005

Epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) are potent mitogenic proteins capable of inducing cell division in a wide variety of cell types. In addition to their mitogenic properties, both proteins have recently been shown to enhance survival and process outgrowth from neurons of central nervous system origin. The full spectrum of neuronal subtypes responding to these factors has not been elucidated. In the present study, EGF was found to enhance survival and process outgrowth of primary cultures of cerebellar neurons of neonatal rat brain. This effect was dose‐dependent and was observed with EGF concentrations as low as 100 pg/ml. In marked contrast, bFGF was ineffective in enhancing survival or neurite elongation from cerebellar neurons when tested in the range of 0.1 to 10.0 ng/ml. However, within this concentration range, bFGF did prove effective in stimulating an increase in [3H]thymidine incorporation into primary cultures of cerebellar astrocytes, demonstrating that bFGF was active and that cells in the cerebellum do respond to bFGF. These results suggest that EGF or an EGF‐like peptide may act as a neurite elongation and maintenance factor for cerebellar neurons. EGF has now been shown to support striatal, cortical, and cerebellar neurons, suggesting that this factor may have trophic activity throughout the central nervous system. bFGF, in contrast, appears to exert its effects on limited populations of neurons.
Distribution of the mineralocorticoid and the glucocorticoid receptor mRNAs in the rat hippocampus
Authors: Van Eekelen J. A. M. , Jiang W. , De Kloet E. R. , Bohn M. C.
Published: January 2005

The cellular localization of mineralocorticoid receptor (MR) and glucocorticoid receptor (GR) gene expression in the rat hippocampus was studied by in situ hybridization using 35S‐labeled RNA‐probes, complementary to either 513 bases of the rat brain mineralocorticoid receptor (MR)‐mRNA or 500 bases of the rat liver glucocorticoid receptor (GR)‐mRNA.

Neurons in CA1, CA2, and the dentate gyrus expressed both receptor genes at high levels. The MR‐mRNA was demonstrated in all pyramidal cell fields (CA1–4) of the hippocampal formation and the granular neurons of the dentate gyrus. In contrast, GR‐mRNA was mainly restricted to CA1 and CA2 pyramidal cell fields and the dentate gyrus. This pattern of hybridization was found to agree with the cellular distribution of the two types of corticosteroid receptors detected previously in the hippocampus by autoradiography of the radio‐labeled receptors and by immunocytochemistry of the receptor protein. These observations suggest that the corticosteroid receptors described previously as type 1 and type 2 are encoded by MR‐ and GR‐mRNA, respectively.

Although both the MR and GR genes are co‐expressed in some hippocampal neurons, the unique patterns of distribution of the two receptor mRNAs in the hippocampal formation suggest that the genes for these receptors are differentially regulated. Moreover, the microanatomy of MR and GR expression provides insight into molecular mechanisms underlying the characteristic action of various steroids on behaviors involved in stress and circadian regulation.
Simple method for the culture of glial cells from embryonic rat brain: Implications for regional heterogeneity and the radial glial lineage
Authors: Wilkie M. B. , Lauder J. M.
Published: January 2005

A simple method is described for the production of glial cell cultures from specific regions of the day 13–15 embryonic rat brainstem and midbrain based on differential cell attachment to a relatively nonadhesive substrate, which inhibits the growth of neurons. Regional differences in the ability of specific populations of brainstem and midbrain cells to attach and spread on the substrate suggest that embryonic glial populations may differ in their cell surface properties even when they derive from the same general area of the developing brain. Based on observations of the spatiotemporal distribution of radial‐like glial cells and astrocytes with time in vitro, we suggest that this culture system may prove useful for investigation of the radial glial lineage.
Developmental expression of neural cell type‐specific mRNA markers in the myelin‐deficient mutant rat brain: Inhibition of oligodendrocyte differentiation
Authors: Kumar S. , Gordon M. N. , Espinosa de los Monteros M. A. , de Vellis J.
Published: January 2005

We have studied gene expression of neuroglial cell markers in the myelin‐deficient (md) rat brain during postnatal development. Northern blots and slot blots of poly(A)+ RNA from developing brain were sequentially probed with cDNAs specific for the oligodendrocyte markers glycerol phosphate dehydrogenase (GPDH), myelin basic protein (MBP), and proteolipid protein (PLP), for the neuronal marker glutamic acid decarboxylase (GAD), and for the astrocyte markers glial fibrillary acidic protein (GFAP) and glutamine synthetase (GS). GPDH mRNA levels were also examined in two peripheral tissues, liver, and skeletal muscle (hindlimb).

Despite a lack of CNS myelin in the md mutant, transcripts of all oligodendroglial markers were detectable except the 1.6‐kb PLP message. Brain GPDH mRNA levels were initially equivalent in md and unaffected littermates at postnatal day 15 (PI5), but the mutants failed to display the normal developmental increase in gene expression. By P25, GPDH mRNA expression in md rat brain was approximately 20% of control levels. GPDH mRNA expression in peripheral tissues was less affected than in brain and was lower in md mutants only at the later developmental ages. Expressions of GAD, GFAP, and GS mRNAs in developing md rat brain were not altered. The mRNA levels of the two myelin markers, MBP and PLP, were severely impaired in md rat brain during the entire myelinating period and represented less than 10% of control mRNA levels at P25. The most important observation was that the large PLP transcript (3.2 kb) was slightly shorter in size in md rat brain as compared to normals. These data indicate that the md mutation does not affect the normal development of astrocytes or GABAergic neurons but severely inhibits the normal differentiation of oligodendrocytes, possibly because of an alteration in the PLP mRNA.
Development of identified glia that ensheathe axons in Hirudo medicinalis
Authors: Morrissey A. M. , McGlade‐McCulloh E.
Published: January 2005

Interaction between neurons and glia may contribute to the formation of characteristic nerve bundles formed by axon elongation along stereotypic pathways. This study reports the temporal and spatial distribution of identified ensheathing glia during embryonic development in the leech. The development of connective glia was followed (1) using an immnohistochemical probe (monoclonal antibody Lan3–13), which recognized connective glia, and (2) using electron microscopy. Embryonic glia were initially located in the medial region of the lateral connectives and contained intermediate filaments. Glia cells continued to develop throughout embryogenesis; the number and size of glial processes increased, and they ensheathed smaller bundles of axons. The glial cell recognized by Lan3–13 first appeared after axons had already begun to form the connectives. This suggests that these particular glial cells may not function in the initial guidance of axons along stereotypic pathways. However, another cell that contained small bundles of intermediate filaments and glycogen granules was present at early stages of connective formation. These cells may be undifferentiated or transient glia, which could contribute to the formation of characteristic nerve bundles.
Anti‐200 kDa neurofilament antibody cross‐reacts with microtubule‐associated protein‐2 (MAP‐2)
Authors: Allende M. L. , Krauss R. Y. , Tremblay C. , Alvarez J. , Inestrosa N. C.
Published: January 2005

An antibody prepared against purified 200‐kDa neurofilament (NF) subunit was found to cross‐react with a high molecular weight polypeptide present in rat brain cytoskeletal extracts starting from early postnatal development. This polypeptide was found to be highly concentrated in microtubule preparations and was specifically recognized as the 280‐kDa microtubule‐associated protein‐2 (MAP‐2). Our results, besides suggesting the existence of common epitopes between the heavy NF subunit and MAP‐2, suggest the possibility of an interaction between two of the most important neuronal cytoskeletal organelles: NFs and microtubules.
Protein kinase C activators reduce the inositol trisphosphate‐Induced outward current and the CA2+‐activated outward current in identified neurons of aplysia
Authors: Sawada M. , Ichinose M. , Maeno T.
Published: January 2005

Effects of intracellularly injected activators of protein kinase C on the InsP3‐induced K+current and the Ca2+‐activated K+current recorded from identified neurons (R9—R12) ofAplysia kurodaiwere investigated with conventional voltage‐clamp and pressureinjection techniques. Intracellular injection of InsP3into identified neurons produced a 4‐aminopiridine (4‐AP)‐resistant, tetraethylammonium (TEA)‐sensitive, and quinidine‐sensitive K+current similar to the Ca2+activated K+current elicited by direct injection of Ca2+ions into the same neurons.

The diacylglycerol analogue 1,2‐oleoylacetylglycerol (OAG) at an intracellular concentration of 65 nM produced irreversible decreases in both the InsP3‐induced K+current and the Ca2+‐activated K+current. The phorbol 12,13‐dibutyrate (PDBu) at an intracellular concentration of 150 nM also decreased irreversibly both the InsP3‐induced K+current and the Ca2+‐activated K+current. These results suggest that protein kinase C activators reduce both the InsP3‐induced K+current and the Ca2+‐activated K+current recorded from certain identified neurons ofAplysiaand that protein kinase C reduces the ability of Ca2+to open K+channels rather than affecting the ability of InsP3to release Ca2+from intracellular stores.
Modulation of acetylcholine release by nicotinic receptors in the rat brain
Authors: De Sarno P. , Giacobini E.
Published: January 2005

Since physostigmine (Phy) is presently used in the experimental treatment of Alzheimer disease (AD) patients by means of intracerebral ventricular (i.c.v.) administration, we designed a study to determine the effect of the drug administered by the same route on the cholinergic system of the rat brain. Particularly, we studied the involvement of nicotinic cholinergic function. The specific conditions required in this experiment were achieved by a series of short‐lasting periods of acetylcholinesterase (AChE) inhibition leading to short‐lasting increases of acetylcholine (ACh). These were produced by periodic i.c.v. injections of Phy. At 7 days of Phy administration, a small effect on 3H‐nicotine binding was seen only in the striatum of the injected side. In rats treated for 13 days, we observed a 120% increase in the stimulated release of 3H‐ACh in hippocampal slices of the injected side of the brain. There also was a significant 88% increase in 3H‐nicotinic binding in the hippocampus of the same side while muscarinic binding was unchanged. These results suggest a process of upregulation of presynaptic nicotinic autoreceptors in the hippocampus modulating ACh release but no effect on the muscarinic receptors. Our results also suggest that pulses of ACh in analogy to nicotinic stimulation can cause protracted desensitization and eventually inactivation of the receptor leading to its up‐regulation. These results are consistent with findings on the release of ACh from cortical biopsies and of a sustained ACh release in the CSF of AD patients following the same treatment.
Conditioned media of regenerating fish optic nerves modulate laminin levels in glial cells
Authors: Cohen A. , Schwartz M.
Published: January 2005

Adult injured optic nerves of mammals show an increased distribution of laminin in the extracellular matrix after application of soluble substances in the form of conditioned media originating from growing nerves (Zak, et al., 1987). This increase could result from direct or an indirect activation of glial cells or from activation of other cellular elements. In the present study, we show that the conditioned media derived from regenerating fish optic nerves contain factors that directly modulate the level of laminin in C‐6 glioma cells. The identity of the laminin was confirmed by metabolic labeling of the treated cells with [35S]methionine and by subsequent immunoprecipitation and gel electrophoresis. The level of other extracellular matrix proteins, such as fibronectin, is also increased. The significance of the presence of glialactivating substances in the conditioned media of regenerating nerves for the process of regeneration is discussed.
Inhibition of the synthesis of glycosphingolipids affects the translocation of proteolipid protein to the myelin membrane
Authors: Pasquini J. M. , Guarna M. M. , Besio‐Moreno M. A. , Iturregui M. T. , Oteiza P. I. , Soto E. F.
Published: January 2005

Brain slices obtained from young rats were incubated with different radioactive precursors, in the presence and absence of L‐cycloserine (an inhibitor of the synthesis of sphingosine) in order to explore the possibility that transport of proteolipids—and specifically of the major myelin proteolipid PLP—to the myelin membrane could be coupled to the transport of cerebrosides or sulfatides. At a concentration of 0.15 mM L‐cycloserine, the incorporation of [3H] glycine into total proteins, proteolipid apoproteins (APL), PLP, and myelin basic proteins (MBP) of the total homogenate was unaffected by the presence of the inhibitor, whereas the incorporation of [3H] serine into glycosphingolipids decreased markedly. Under similar incubation conditions, the entry of labeled APL and of PLP into the myelin membranes in the presence of L‐cycloserine decreased markedly (50%) in comparison to controls. Entry of MBP was not affected by the inhibitor. These results indicate that when synthesis of glycosphingolipids is inhibited by L‐cycloserine, thus decreasing the availability of cerebrosides and sulfatides, the translocation of PLP to myelin is disrupted, suggesting that its transport through the oligodendroglial cell could be coupled to the transport of glycosphingolipids and, most probably, of sulfatides.
Pre‐ and post‐natal ontogeny of serotonergic projections to the rat spinal cord
Authors: Rajaofetra N. , Sandillon F. , Geffard M. , Privat A.
Published: January 2005

The development of 5‐hydroxytryptamine (5‐HT) innervation in the spinal cord was studied from embryonic day 14 (E14) to adulthood. Sprague‐Dawley rats were fixed by perfusion with 5% glutaraldehyde in cacodylate‐sodium metabisulfite buffer, and vibratome sections were processed for immunocytochemistry with a 5‐HT antiserum. For electron microscopy, the sections were flat‐embedded in araldite, and thin sectioning was performed. 5‐HT neurons caudally directed from raphe nuclei invade the spinal cord at E14 and reach the caudalmost levels by E16‐E17. In longitudinal sections, axons are seen by E15, at cervical and upper thoracic levels, to invade the presumptive gray matter from the anterior and lateral funiculi. The invasion process occurred either by sharp angulation of the axon or by branching of a collateral. By E16, at thoracic level the anterior horn and the intermediolateral columns are profusely innervated by very thin, varicose fibers; synapses are seen at E17 and E18 using EM. 5‐HT immunoreactive boutons are involved here. After birth, 5‐HT innervation of these two areas evolves progressively from a diffuse network to a more restricted pattern, especially at the thoracic level for the intermediolateral column and at cervical and lumbar levels for the anterior horn. The adult pattern is reached by postnatal day 21 (P21). The growth of axons toward the dorsal horn becomes noticeable by E19 at all spinal levels, when fibers invade the neck of the horn from the lateral funiculus, and innvervation proceeds diffusely until P5. At P7, thin fibers course dorsally and laterally along the border of the gray matter and ramify profusely in layers I and II. The adult pattern is also reached in the dorsal horn by P21. These results are discussed in relation to the postnatal maturation of motor and sensory circuits and to the development of transplanted raphe neurons in the rat spinal cord.
Developmental and regional expression of three new members of the ras‐gene family in the mouse brain
Authors: Ayala J. , Olofsson B. , Touchot N. , Zahraoui A. , Tavitian A. , Prochiantz A.
Published: January 2005

We have examined the expression in the mouse nervous system of three new members of the ras protooncogene family: rab1, rab2, and rab3. Each of these genes was transcribed into messenger RNAs with different molecular weights. These transcripts had specific developmental and regional patterns of expression. In particular, for the three genes, the ratio between the heavy and light mRNAs depended strongly on developmental stage and brain region. The use of pure neuronal and glial cultures revealed that the high molecular weight transcripts were enriched in neurons and that, in the case of rab2 and rab3, their expression increased with neuronal differentiation. These results are discussed considering the sequence identities between these genes and the yeast YPT1 and sec‐4 genes, which are known to be implicated in post‐Golgi vesicular transport and cytoskeletal stabilization. We propose that the rab genes might be of importance in the regulation of these two processes within the developing and adult nervous system.