bluuebunny/crossref_metadata_2025 huggingface dataset:
Expression of an engrailed‐like gene during development of the early embryonic chick nervous system
Authors: Gardner C. A. , Darnell D. K. , Poole S. J. , Ordahl C. P. , Barald K. F.
Published: January 2005

The engrailed gene has been identified in Drosophila as an important developmental gene involved in the control of segmentation. Here we describe the embryonic expression of a chicken gene, ChickEn (Darnell et al.: J Cell Biol 103(5):311a, 1986), which contains homology to the Drosophila engrailed gene. Northern blots of early chick embryo tissue poly(A)+ RNA resulted in hybridization to at least three bands expressed predominantly in the brain/head region when probed with ChickEn genomic fragments. Eight cDNA clones generated from embryonic day 6 (stage 29–30) chick brain poly(A)+ RNA are identical in their nucleotide sequence with the ChickEn genomic clone. In situ hybridization to sections of 4‐day (stage 24) embryos indicated that ChickEn transcripts were concentrated in the posterior mesencephalon and anterior metencephalon. In cultures of chick cranial neural crest cells (eight to nine somites; stage 9) ChickEn transcripts were localized in a subset (approx. 8%) of cells examined after 2 days in culture.

A mouse monoclonal antibody, inv‐4D9D4, made by Coleman and Kornberg recognizes the engrailed‐like homeo domain of the engrailed and invected proteins (Martin‐Blanco, Coleman, and Kornberg, personal communication). Patel, Coleman, Kornberg and Goodman (unpublished) have shown that this antibody binds to the hindbrain of 2‐day‐old chick embryos. We have confirmed these results and shown that this antibody binds to the same region of 4‐day (stage 24) chick brains that in situ hybridization showed contained ChickEn transcripts. This antibody also recognizes a homeo domain‐containing ChickEn peptide expressed as a beta‐galactosidase fusion protein in Drosophila cell culture. We have not detected ChickEn protein in any tissue prior to eight to nine somites (stage 9). These results delineate the major expression pattern of the ChickEn gene during early (prior to stage 30) embryonic development in the chick.
Human fetal schwann cells in culture: Phenotypic expressions and proliferative capabibility
Authors: Kim S. U. , Yong V. W. , Watabe K. , Shin D. H.
Published: January 2005

In this report we examined the phenotypic expressions and the proliferative capability of cultured human fetal Schwann cells. Antigens that were expressed included laminin, nerve growth factor receptor, neural cell adhesion molecule, S‐100 protein, and that recognized by the monoclonal antibody HNK‐1. In addition, HLA‐A,B,C and HLA‐DR, respectively, class I and class II antigens of the major histocompatibility complex, were demonstrated on Schwann cells. Mitotic capability was high, with an average of 34% of Schwann cells undergoing proliferation over a 2‐day period.
Identification of a cDNA clone specific for the neural cell adhesion molecule AMOG
Authors: Pagliusi S. , Antonicek H. , Gloor S. , Frank R. , Moos M. , Schachner M.
Published: January 2005

A cDNA clone of the neural cell adhesion molecule AMOG was isolated from a lambda gt10 library constructed from 8‐day‐old mouse brain poly(A) + RNA with a 17mer oligonucleotide probe designed from a nonapeptide sequence obtained from tryptic peptides of AMOG. The cDNA clone expressed as a fusion protein that is recognized by polyclonal AMOG antibodies; conversely, polyclonal antibodies prepared against the fusion protein react with AMOG. The clone contains the full sequence derived from the nonapeptide. Of all tissues tested, only brain expresses detectable levels of AMOG by ELISA and Northern blot analyses, indicating a high correlation in expression at the protein and mRNA levels. Both brain and astrocytes express a 3 kb long mRNA, which appears to be encoded by a single gene.
Adult retinal ganglion cells retain the ability to regenerate their axons up to several weeks after axotomy
Authors: Thanos S. , Vanselow J.
Published: January 2005

The present work was to elucidate whether the ability of adult central neurons to regrow their lesioned axons is retained for long periods of time. Using the rat retina as an experimental paradigm, the optic nerve was lesioned by crush in situ. Up to 6 weeks after the trauma, the optic nerve (ON) was again exposed and transected close to the first lesion and autologous sciatic nerve segments were anastomosed at the ocular ON stump. Alternatively, the retina corresponding to the lesioned ON was dissected for in vitro cultivation 1–6 weeks after the crush‐axatomy was applied. Both experimental strategies revealed a regrowth of the lesioned retinal ganglion cell (RGC) axons. When peripheral nerve (PN) segments were grafted without previous ON crush, axon stumps started to reelongate and to penetrate the grafted piece of nerve 6 days later. In contrast, when the PN graft was apposed to the ON stump 1–6 weeks after crush, it was penetrated by regrowing axons within 24 hr. Maximal numbers of regenerating axons were observed if transplantation occurred within the first week after the crush. The numbers of axons decreased progressively if transplantation was performed later than 1 week postcrush and approximated zero values at the 6th week. When the noncrushed retina was explanted and cultured in vitro in a chemically defined, serumfree medium, there was almost no extending fiber. In contrast, explantation of the retina for which the ON had been precrushed in situ resulted in massive regrowth of RGC axons. The numbers of regenerating axons and their temporal changes paralleled those described for the transplantation experiments. The data indicate that axomotized central neurons sustain their ability for axon regrowth over considerable periods of time and can make use of this elongation capability if regeneration‐permissive environmental conditions are provided.
Effect of cocaine and cocaine congeners on veratridine‐induced depolarization in mouse cerebrocortical synaptoneurosomes
Authors: Zimányi I. , Wang E. , Lajtha A. , Reith M. E. A.
Published: January 2005

Structure‐activity relationships were determined for cocaine congeners in counteracting the depolarization induced by the action of veratridine on voltage‐dependent sodium channels of synaptoneurosomes from mouse brain cortex, and their potency was compared to those determined previously on Na+ uptake and batrachotoxinin binding. Cocaine, norcocaine, (+)‐pseudococaine, (‐)‐pseudococaine, (+)‐neopseudococaine, benzoyltropine, benzoylpseudotropine, ecgonine methylester, atropine, WIN‐35,428, WIN‐35,140, WIN‐35,065‐3, WIN‐35,004, and procaine were tested for their potency in inhibiting depolarization as measured by the distribution of the lypophilic cation [3H]triphenylmethylphosphonium across the membrane. All of the tested compounds inhibited the veratridine‐induced depolarization in a competitive manner. The structure‐activity relationships were similar to those for inhibition of 22Na+ uptake in mouse brain homogenates, and the potency of these local anesthetics in inhibiting veratridine‐induced uptake of [3H]triphenylmethylphosphonium correlated well with their potency in inhibiting [3H]batrachotoxinin A 20‐α‐benzoate binding in mouse brain synaptosomes.
Differentiation‐regulated loss of the polysialylated embryonic form and expression of the different polypeptides of the neural cell adhesion molecule by cultured oligodendrocytes and myelin
Authors: Trotter J. , Bitter‐Suermann D. , Schachner M.
Published: January 2005

The expression of the neural cell adhesion molecule (N‐CAM) on cultured murine oligodendrocytes, their precursors, and myelin was examined by indirect immunofluorescence, biosynthetic radiolabeling followed by immunoprecipitation and Western blot analysis, using antibodies specific for various forms of the molecule. In all culture systems studied, whether the oligodendrocytes were cultured as an enriched fraction containing precursor cells or in the presence of astrocytes and neurons, a similar differentiation stage‐related expression of N‐CAM was seen. At early developmental stages many tetanus toxin receptor and A2B5 antigen‐positive putative oligodendrocyte precursors with bipolar morphology were seen and found to express N‐CAM in its embryonic form. Of the 04 antigen‐positive immature oligodendrocytes with few slender processes most expressed N‐CAM, but few the embryonic form of N‐CAM. The more mature 01 or 010 antigen‐positive oligodendrocytes were found to express exclusively the adult form of N‐CAM. Oligo‐dendrocytes synthesized the 120 and 140 kD forms of N‐CAM (N‐CAM 120 and N‐CAM 140), but not N‐CAM 180, although with differentiation, N‐CAM 120 predominated in oligodendrocytes and also in pure myelin. N‐CAM 120 could be released from oligodendrocytes and myelin by phosphatidylinositol‐specific phospholipase C, suggesting that in both oligodendrocytes and myelin N‐CAM 120 is inserted into the membrane by covalent linkage to phosphatidylinositol.
Neurotransmitter phenotype plasticity in cultured dissociated adult rat dorsal root ganglia: An immunocytochemical study
Authors: Schoenen J. , Delree P. , Leprince P. , Moonen G.
Published: January 2005

Culturing sympathetic ganglion neurons in vitro may modify phenotypic expression of some neurotransmitters. For dorsal root ganglia (DRG), contradictory results have been reported; most studies have used immature material. We have therefore performed a detailed immunocytochemical analysis of the transmitter content of cultured adult rat DRG neurons. To demonstrate possible modifications of neurotransmitter phenotypes, we have compared the results obtained with the same techniques on neurons cultured for 3 days and on freshly dissociated DRG cells. Also, the transmitter profile of cultured neurons was compared with that known from in situ studies.

Out of 22 antigens studied, 20 were detected in cultured DRG neurons. All of them were expressed in small and/or intermediate‐sized cells. Large neurons only contained CGRP, VIP, NPY, β‐END, ENK, and GABA. The percentage of immunostained neurons varied for the various antisera: < 10% of cultured neurons were positive for ENK, β‐LPH, β‐END, DYN, VASO, and OXY; 10–30% for SOM, CCK, CAT, and SP; and > 30% for NPY, CRF, GLU, NT, VIP, GABA, GRP, CGRP, 5‐HT, and TRH. In the latter two groups of transmitters (except CGRP), the proportion of immunoreactive neurons was by far larger in cultured than in freshly dissociated DRG. The most pronounced ( > 25%) increase in the proportion of positively stained neurons after culturing was observed for the GRP, CRF, TRH, and 5‐HT antisera. Serotonin was the only transmitter identified in cultured but not in freshly dissociated cells.

These data indicate, on one hand, that various antigens, for example, CAT, GABA, NT, TRH, NPY, β‐LPH, and β‐END, which up to now have not been described in DRG in situ, can be detected immunocytochemically a few hours after dissociation of adult rat DRG. On the other hand, several transmitters, for example, VIP, NPY, SP, GABA, GLU, NT, GRP, CRF, TRH, and 5‐HT, are expressed in a significantly higher proportion of cells in cultured than in freshly dissociated preparations. This might reflect a change in the phenotypic expression of transmitters due to the new environment generated by the culture conditions, a hypothesis that can be tested by measuring specific mRNA levels. Moreover, considering the plasticity and multipotentiality of their transmitter phenotype, cultured adult DRG neurons might represent an interesting material for autografts into the injured central nervous system.
Isolation and structural characterization of the bovine tyrosine hydroxylase gene
Authors: D'Mello S. R. , Turzai L. M. , Gioio A. E. , Kaplan B. B.
Published: January 2005

A bovine tyrosine hydroxylase (TH) cDNA probe was used to screen a charon 30 genomic library. Screening of approximately 1 million recombinant phage resulted in the identification of one clone, γB1, containing the entire bovine TH gene. Results derived from restriction endonuclease mapping and sequence analysis reveal that the bovine gene contains 13 exons spanning approximately 7 kb of genomic DNA. Determination of the transcription initiation site indicates that the TH gene has a 5′ untranslated region of 27 bp. A TATA‐box sequence is located between positions −29 and −24 from the transcription initiation site and a cyclic AMP regulatory element (CRE) between −45 and −38. Although the TH gene appears to be glucocorticoid responsive in vitro, no regions bearing identity to the consensus sequence for the glucocorticoid regulatory element (GRE) were detected within approximately 1.5 kb of 5′ flanking sequence. A cross‐species comparison of the 5′ flanking sequences of the bovine, rat, and human TH genes reveals strong sequence and positional conservation of seven sequence elements. An analysis of the nucleotide sequence within these elements reveals similarity to the consensus sequences reported for known cis‐acting regulatory elements and transcription factor binding sites, suggesting that they may play a role in the regulation of TH gene expression.
Identification of basic fibroblast growth factor as a cholinergic growth factor from human muscle
Authors: Vaca K. , Stewart S. S. , Appel S. H.
Published: January 2005

Dissociated embryonic chick ciliary ganglion cells in culture were used as a bioassay to isolate a cholinergic growth‐promoting protein from extracts of autopsied adult human muscle. An active protein was purified after acid and salt precipitation of extract, cation exchange, molecular sieving, heparin affinity chromatography, and in some cases, SDS‐PAGE. This protein increased levels of choline acetyltransferase activity and ACh synthesis with time in culture. The protein was identified as basic FGF by several criteria. It shared the high affinity for heparin and was the same approximate molecular weight, 18 kD, as basic FGF. Activity was removed from solution by antibodies specific for basic FGF. Recombinant human basic FGF was equally effective in stimulating CAT activity, but was not additive with our purified protein at saturating concentrations. Basic FGF was also found in extracellular matrix and conditioned medium from cultured embryonic chick muscle. The activity could be released from extracellular matrix by treatment with heparinase or high salt extraction. Basic FGF stimulates neurite outgrowth as well as the capacity for transmitter synthesis. Thus, basic FGF is present in embryonic and adult muscle and capable of acting as a growth regulator for cholinergic neurons.
Sodium‐independent transport of noradrenaline in mouse and rat astrocytes in primary culture
Authors: Paterson I. A. , Hertz L.
Published: January 2005

The uptake of noradrenaline by primary cultures of mouse and rat astrocytes was investigated in order to examine whether an inhibition of extraneuronal noradrenaline uptake was the mechanism whereby some trace biogenic amines potentiate neuronal responses to noradrenaline. In the presence of inhibitors of the enzymes monoamine oxidase and catechol‐O‐methyl transferase, it was found that astrocytes took up noradrenaline by a temperature‐dependent, sodium‐independent mechanism that was saturable with a Km = 3.4 × 10−7 M and a Vmax = 1.6 pmole/mg protein/2 min. This uptake mechanism did not concentrate noradrenaline within the cell. The uptake of noradrenaline was inhibited by ascorbic acid (IC50 = 3.4 × 10−7 M), adrenaline (IC50 = 7.9 × 10−7 M), and dopamine (IC50 = 1.5 × 1.0−6 M). It was not inhibited by the tricyclic antidepressants amitriptyline and desmethylimipramine or the trace biogenic amines β‐phenylethylamine, phenylethanolamine, p‐ and m‐tyramine and p‐ and m‐octopamine. Nor was the uptake inhibited by fluoxetine or 5‐hydroxytryptamine. It is concluded that astrocytes take up noradrenaline by a facilitated‐diffusion mechanism and that this uptake resembles the extraneuronal uptake described in preparations of brain tissue. It is also concluded that the trace biogenic amines do not potentiate neuronal responses to noradrenaline by inhibiting extraneuronal uptake.
Changes in free fatty acids, phospholipids, and cholesterol following impact injury to the rat spinal cord
Authors: Demediuk P. , Daly M. P. , Faden A. I.
Published: January 2005

Free fatty acids (FFA), phospholipid, and cholesterol levels were measured in spinal cord samples from rats subjected to low (25 g‐cm), moderate (50 g‐cm), or severe (100 g‐cm) impact trauma to the T10 spinal segment. All degrees of injury caused early (15 min) declines in total phospholipids after trauma; phospholipid levels remained significantly below controls in rats subjected to moderate and severe injuries for up to 3 days, whereas phospholipids had returned to baseline values by 4 hr in the low injury group. Rapid and persistent decreases in cholesterol levels were observed for all injury groups. Severe trauma was associated with biphasic increases in FFA levels: levels were elevated at 5 and 15 min post‐trauma and had declined by 30 min; a second elevation was observed at 1 hr, progressively increasing to reach a maximum at 24 hr, before declining over the next 6 days. Low and moderate injuries caused similar early total FFA increases; later increases were significantly smaller than in the severely injured group. Among the free fatty acids, significant increases were observed in palmitate, stearate, oleate, linoleate, linolenate, arachidonate, and docosahexaenoate. These findings indicate that traumatic spinal cord injury results in early, transient, postinjury membrane phospholipid hydrolysis, the magnitude of which is relatively independent of the severity of injury. More delayed and sustained lipid hydrolysis also occurs after trauma, the magnitude of which is related to the severity of injury.
Oligodendrocytes express a normal phenotype in carbonic anhydrase II‐deficient mice
Authors: Ghandour M. S. , Skoff R. P. , Venta P. J. , Tashian R. E.
Published: January 2005

The nervous system of a mouse mutant characterized by a carbonic anhydrase II (CA II) deficiency was examined with light and electron microscopy and with immunocytochemistry using different glial cell markers.

No major morphologic abnormalities at either the cellular or subcellular level are detectable in the brains of CAII‐deficient mice, even though CAII is the main isozyme of CA in the brain. The oligodendrocytes, which characteristically express high levels of CA II, do not exhibit signs of degeneration or abnormalities even in 1‐year‐old CA II‐deficient mice. Similarly, neurons and astrocytes have a normal structure and distribution. Oligodendrocytes show a normal staining pattern and distribution for galactocerebroside (GC), 2′, 3′‐cyclic nucleotide 3′‐phosphohydrolase (CNP), and myelin basic protein (MBP). Astrocytes have a normal morphology and distribution when stained for GFAP and S100 protein. The lack of major degeneration in the brain due to a CA II deficiency suggests these mice utilize other enzymatic or physiological pathways to compensate for the enzyme absence.
Neurotrophic activity of monomeric glucophosphoisomerase was blocked by human immunodeficiency virus (HIV‐1) and peptides from HIV‐1 envelope glycoprotein
Authors: Mizrachi Y.
Published: January 2005

Glucophosphoisomerase (GPI), a glycolytic enzyme, was recently described to share 90% sequence homology with neuroleukin, a recently discovered growth factor which promotes motor neuron regeneration in vivo, survival of peripheral and central neurons in vitro, and affects B cell immunoglobulin synthesis.

Interestingly, neuroleukin activity was described to be antagonized by the human immunodeficiency virus (HIV‐1) envelope glycoprotein (gp120), with which neuroleukin was found to share partial sequence homology. In this study, reduced GPI demonstrated similar activity to neuroleukin in a novel bioassay using human and rat neuroblastoma cell lines. In the presence of reduced GPI, these cells were found to differentiate, in terms of enhanced neurite extension at a reduced proliferation rate. These results demonstrate the existence of a novel growth factor activity of an evolutionary ancient enzyme. The nonreduced commercial form of GPI, probably the dimer, was found to be inactive in this bioassay. Using the neuroblastoma cells model system, we further investigated the significance of the region of homology to HIV‐1 envelope glycoprotein (gp120) as the putative binding site of GPI to its receptor on neuronal cells.
Complete sequence of a cDNA encoding an active rat choline acetyltransferase: A tool to investigate the plasticity of cholinergic phenotype expression
Authors: Brice A. , Berrard S. , Raynaud B. , Ansieau S. , Coppola T. , Weber M. J. , Mallet J.
Published: January 2005

A cDNA clone encoding the complete sequence of an active, rat choline acetyltransferase (ChoAcTase; acetyl‐CoA:choline O‐acetyltransferase, EC 2.3.1.6) has been isolated. Analysis of the deduced amino acid sequence reveals 85% and 31% identity with the porcine and Drosophila melanogaster enzymes, respectively. To further elucidate the molecular basis of neurotransmitter‐related phenotypic plasticity, the expression of ChoAcTase mRNA was compared with that of tyrosine hydroxylase [TH; tyrosine 3‐monooxygenase, L‐tyrosine, tetrahydropteridine: oxygen oxidoreductase (3‐hydroxylating), EC 1.14.16.2], in neurons from superior cervical ganglia grown in the following conditions: (1) normal medium, (2) high K+ medium, and (3) normal medium supplemented with 50% muscle‐conditioned medium (CM). TH mRNA was expressed in all three media; its level rose in high K+ and decreased strikingly in the presence of CM. ChoAcTase mRNA could be visualized in CM, but fell to undetectable levels in normal and high K+ media. These results suggest that translational or post‐translational mechanisms do not play a major role for the modulation of neurotransmitter‐associated phenotype.
Quantitative analysis of the localization of adrenergic binding sites in chick brain
Authors: Dermon C. R. , Kouvelas E. D.
Published: January 2005

In the present work [3H]‐WB4101, [3H]‐DHA, and [3H]‐clenidine were used for the study of the localization of α1, α2, and βT adrenergic receptors in the chick brain.

The highest concentration of [3H]‐WB4101 was observed in the nucleus pretectalis, followed by the nucleus brachium conjuctivum descendens. The supercial layers of stratum griseum fibrosum superficiale, the nucleus mesencephalis lateralis pars dorsalis, and the locus coeruleus showed concentrations of [3H]‐WB4101 binding higher than 300 fmoles/mg protein. Concentrations of [3H]‐DHA binding higher than 300 fmoles/mg protein were observed in the paleostriatum, the external part of nucleus pretectalis, the nucleus isthmi parvocellularis, the nucleus mesencephalis lateralis pars dorsalis, the dorsal nucleus of oculomotor center, and the molecular layer of cerebellum. Locus coeruleus was the only area of chick brain which showed concentration of [3H]‐clonidine binding higher than 300 fmoles/mg protein. With few exceptions, [3H]‐clonidine binding was very low and in the telencephalon it was undetectable.
Heme precursor 5‐aminolevulinic acid alters brain tryptophan and serotonin levels without changing pineal serotonin and melatonin concentrations
Authors: Daya S. , Nonaka K. O. , Buzzell G. R. , Reiter R. J.
Published: January 2005

The daytime administration of the heme precursor 5‐aminolevulinate (5‐ALA) has been shown to reduce brain tryptophan and serotonin levels owing to saturation of liver tryptophan pyrrolase. Saturation of this enzyme with heme results in enhanced activity, leading to increased catabolism of tryptophan and thus making less tryptophan available to the brain. Tryptophan is also the precursor of melatonin, a primary secretory product of the pineal gland. Reduced melatonin levels, which could be a consequence of altered tryptophan metabolism, have been associated with depressive disorders in humans. In view of this, in the present study we examined the effects of 5‐ALA administration on forebrain tryptophan and serotonin levels as well as pineal serotonin, melatonin, and the pineal melatonin‐forming enzymes N‐acetyltransferase (NAT) and hydroxyindole‐O‐methyltransferase (HIOMT). 5‐ALA induced a reduction in forebrain tryptophan and serotonin levels during the light phase and caused the opposite effect in the dark phase. Allopurinol, an inhibitor of hepatic tryptophan pyrrolase activity, prevented the reduction in the indole levels induced by 5‐ALA. Although 5‐ALA induced these changes in the forebrain, it did not alter either daytime or nighttime pineal NAT, HIMOT, serotonin, or melatonin levels. It appears that high serotonin levels and melatonin production in the pineal are conserved even in the face of low circulating tryptophan levels, changes which alter brain tryptophan and serotonin concentrations.
Increase of transferrin receptors and iron uptake in regenerating motor neurons
Authors: Graeber M. B. , Raivich G. , Kreutzberg G. W.
Published: January 2005

After injury, motor neurons exhibit a number of metabolic and protein changes that are assumed to be part of an inherent neuronal regeneration program, which, when activated, eventually leads to functional restitution. The mechanisms underlying this regeneration are unclear, but it may be expected that factors supporting neuronal growth or survival play an important role in the restoration of neuronal integrity. A number of neuronal growth‐associated proteins have been identified, but their functional roles remain unclear. This paper shows that axotomy results in a strong increase in transferrin receptors (TfRs) in regenerating motor neurons and that this phenomenon is functionally associated with an elevated uptake of exogenous iron. The association of TfR expression in regenerating motor neurons with direct uptake of iron into the brain provides evidence that iron uptake into neural tissue may be related to neuronal metabolic activation. We suggest that the enhanced capacity of regenerating motor neurons to bind transferrin and to take up iron plays an important role in neuronal repair.
Stimulation of rat B‐lymphocyte proliferation by corticotropin‐releasing factor
Authors: McGillis J. P. , Park A. , Rubin‐Fletter P. , Turck C. , Dallman M. F. , Payan D. G.
Published: January 2005

The mitogenic effect of corticotropin‐releasing factor (CRF) on rat lymphocytes was investigated. When rat splenocytes were cultured for 48 hr with CFR, a dose‐dependent increase in incorporation of 3H‐thymidine (3H‐Tdr) was observed, with a maximal response at 10 nM CRF. Comparison of the proliferative effect of CRF on enriched populations of B lymphocytes, T lymphocytes, or macrophages revealed that only B lymphocytes responded following treatment with CRF. When lymphocytes derived from different lymphoid tissues were compared, CRF had a greater proliferative effect on lymphocytes derived from gut‐associated lymphoid tissue (mesenteric lymph nodes and Peyer's patches) than on lymphocytes from spleen or inguinal lymph nodes; CRF had no effect on thymocytes. Synthetic fragments of CRF were used to determine which portions of the peptide are recognized by lymphocytes. The C‐terminal fragments α‐helical CRF9–41 and CRF21–41 were as potent as native CRF in stimulating B‐lymphocyte proliferation, whereas CRF1–20 did not stimulate proliferation. The activity of these peptides suggests that CRF stimulates lymphocyte proliferation by cellular recognition of structural determinants in the C‐terminal one‐half of the peptide.
Epidermal growth factor inhibits the expression of myelin basic protein in oligodendrocytes
Authors: Sheng H. Z. , Turnley A. , Murphy M. , Bernard C. C. A. , Bartlett P. F.
Published: January 2005

The major function of the oligodendrocyte is to myelinate axons in the central nervous system (CNS). Two of the components of myelin, galactocerebroside (galc) and myelin basic protein (MBP), have been used as markers of oligodendrocyte maturation in the developing CNS, and it has been found that galc+ cells arise initially, which then mature into MBP+ oligodendrocytes several days later. We have been interested in the control of expression of MBP and have followed its appearance in cultures of brain cells isolated from 4 day‐old mice. In low serum (0.5% foetal bovine serum), approximately 330 MBP+ cells arise per 2 × 105 brain cells after 3 days incubation. We have examined the ability of several growth factors to influence the expression of MBP in these cultures, including epidermal growth factor (EGF), platelet‐derived growth factor (PDGF), and the fibroblast growth factors (acidic and basic FGF). EGF was found to suppress strongly the developmental expression of MBP in these cultures, but the suppression was reversible, since the number of MBP+ cells approached control numbers 3 days after removal of EGF from the cultures. It was also found that MBP could be down‐regulated in mature MBP+ oligodendrocytes. The action of EGF in these cultures could be mimicked by transforming growth factor‐α (TGFα). The effects of EGF appear to be associated primarily with MBP production in oligodendrocytes since expression of galc is unaffected by EGF.
Axonal transport of a clathrin uncoating atpase (HSC70): A role for hsc70 in the modulation of coated vesicle assembly in vivo
Authors: de Waegh S. , Brady S. T.
Published: January 2005

Clathrin plays an important role in many cellular processes, including endocytosis, secretion, and sorting of membranous organelles. Both the neuronal cell body and presynaptic terminals contain numerous coated vesicles, but few are detectable in the axonal regions that connect these two regions of the neuron. Clathrin heavy chains, light chains, and assembly proteins have all been shown to be axonally transported as part of slow component b (SCb). However, the paucity of coated vesicles present in the axon indicates the existence of a mechanism regulating clathrin coated assembly in vivo. A clathrin uncoating ATP‐ase has been described that binds in stoichiometric amounts to clathrin and dissociates clathrin coats from vesicles in the presence of ATP in vitro. This clathrin uncoating ATPase is a major cytosolic protein of Mr 70 kD in bovine brain, forming 1% of soluble brain protein, and appears to be homologous with a constitutively expressed 70 kd heat shock protein (HSC70). We report here that a major 70 kD protein present in the SCb rate component of axonal transport is identical with HSC70 by both biochemical and immunochemical criteria. The cotransport of HSC70 with clathrin in SCb of axonal transport is consistent with a role for HSC70 in vivo in the regulation of clathrin function during axonal transport.
Role of protein tyrosine phosphorylation in the NGF response
Authors: Maher P. A.
Published: January 2005

The role of protein tyrosine phosphorylation in the response of PC12 cells to NGF was investigated by using a variety of agents which affect NGF‐induced neurite outgrowth. K‐252a, a kinase inhibitor, was previously found to selectively inhibit many of the actions of NGF on PC12 cells. In the present study, it was shown to inhibit NGF‐induced protein tyrosine phosphorylation. However, sphingosine, an inhibitor of protein kinase C and NGF‐induced differentiation of PC12 cells, did not alter the phosphorylation of proteins on tyrosine stimulated by NGF. Disruption of either actin microfilaments or microtubules also had no effect on NGF‐induced protein tyrosine phosphorylation in PC12 cells. The effect of vanadate, an inhibitor of phosphotyrosyl phosphatases, on the differentiation of PC12 cells was also examined. Vanadate did not promote neurite outgrowth but did stimulate protein tyrosine phosphorylation. Taken together, these results suggest that protein tyrosine phosphorylation is one of the first events in the NGF pathway in PC12 cells but alone is not sufficient to induce morphological differentiation. Finally, the distribution of phosphotyrosine‐containing proteins in untreated and NGF‐treated cells was examined by immunofluorescence microscopy. The distribution of these proteins was altered by treatment of the cells with NGF and appeared to correlate with the distribution of actin filaments, particularly in growth cones.
Molecular cloning of human myelin‐associated glycoprotein
Authors: Spagnol G. , Williams M. , Srinivasan J. , Golier J. , Bauer D. , Lebo R. V. , Latov N.
Published: January 2005

The nucleotide sequence for human myelin‐associated glycoprotein (MAG) and its deduced amino acid sequence, obtained by analysis of two overlapping cDNA clones isolated from a human brain cDNA library, is presented and compared to that reported for rat MAG.

The sequence provides an open reading frame of 1,878 nucleotides encoding a peptide of 626 amino acids with a calculated molecular weight of 69.1 kD. It is 89% homologous in nucleotide sequence to the large isoform of rat MAG, with 95% homology in the amino acid sequence. It contains 9 potential glycosylation sites, one more than in rat, and shares other key features with rat MAG, including 5 immunoglobulin‐like regions of internal homology, an RGD sequence, and potential phosphorylation sites. Its structure appears to be highly conserved in evolution, possibly suggesting a close interdependence between its structure and function. The human gene is located on the proximal long arm of chromosome 19 (19q12 → q13.2).
Denervation and hyperinnervation in the nervous system of diabetic animals. II. Monoaminergic and peptidergic alterations in the diabetic encephalopathy
Authors: Di Giulio A. M. , Tenconi B. , La Croix R. , Mantegazza P. , Abbracchio M. P. , Cattabeni F. , Gorio A.
Published: January 2005

The monoaminergic innervation of the central nervous system (CNS) is characterized by long and short projecting neurons. The neurological correlates of diabetes are usually referred to as processes of degenerative atrophy affecting motor and sensory peripheral nerves. We have found that the long serotoninergic axons innervating the spinal cord and the cerebral cortex are unaffected in diabetic animals and that the noradrenergic innervation of the cortex is normal as well. The serotonin content is doubled in the hypothalamus with no apparent alteration of 5‐HIAA levels, suggesting a supernumerary innervation that is accompanied by a reduced release. In pons medulla oblongata, serotonin and dopamine with the relative metabolites 5‐HIAA and DOPAC are significantly reduced, whereas noradrenaline is markedly increased. In the hippocampus, there is a reduction of serotonin content. The serotoninergic alterations are peculiar as suggested by the sparing of the most distal projections that is accompanied by hyperinnervation of the hypothalamus and the loss of shorter collaterals in the pons medulla oblongata. In the hypothalamus and in the striatum of diabetic rats, there are significant higher levels of substance P and met‐enkephalin, respectively. The abundance of proenkephalin A mRNA is also increased in the striatum. Conversely, in the lumbar cord of diabetic animals, the levels of substance P and met‐enkephalin are significantly reduced. Such alterations likely reflect retrograde degeneration of the peripheral sensory input. The CNS changes are unlikely due to vascular abnormalities in the brain of diabetic rats; rather, we suggest that the persistent lack of insulin is the major factor involved as a trigger of the monoaminergic changes in the diabetic brain.
Chemical deglycosylation on a micro‐scale of membrane glycoproteins with retention of phosphoryl‐protein linkages
Authors: Horvath E. , Edwards A. M. , Bell J. C. , Braun P. E.
Published: January 2005

We provide here an easy and convenient procedure for the chemical deglycosylation of membrane glycoproteins that can be done rapidly and inexpensively on large numbers of membrane samples on a microscale without prior isolation of the glycoprotein in question. We have used the procedure successfully on three different glycoproteins and additionally have shown that covalent linkages of the polypeptide to phosphoryl residues are preserved.
GM1 Ganglioside treatment after global ischemia protects changes in membrane fatty acids and properties of Na+, K+‐ATPase and Mg2+‐ATPase
Authors: Mahadik S. P. , Hawver D. B. , Hungund B. L. , Li Y. S. , Karpiak S. E.
Published: January 2005

An examination was made of the effects of ganglioside GM1 (i.m.) on the losses of membrane fatty acids (palmitic, stearic, oleic, linoleic, and arachidonic), the plasma membrane enzyme Na+, K+‐ATPase, and the mitochondrial membrane enzyme Mg2+‐ATPase, associated with global ischemia 24 hr after permanent unilateral occlusion of the carotid artery in Mongolian gerbils. While there was a significant loss of fatty acids in saline controls, no loss was detected in membranes from GM1‐injected gerbils. Rather, we found an increase in membrane fatty acid content, indicative of altered turnover. A 38% loss of Na+, K+‐ATPase and a 36% loss of mitochondrial Mg2+‐ATPase observed in membranes from saline controls was reduced in membranes from GM1‐injected animals to losses of 15% and 8% respectively. These effects are further described by analyses of enzyme kinetics (apparent Vmax and apparent Km). After 1 week of storage, the activities of both membrane ATPases from saline controls decreased substantially more than from GM1‐injected animals, suggesting that the GM1 membranes were better “preserved.” Since there was a minimal loss in protein content after 24 hr of ischemia, these results indicate that systemically injected GM1 may protect structure and function of plama membranes during the acute phases of ischemic injury.
Free radicals mediate peroxidative damage in guinea pig hippocampus in vitro
Authors: Pellmar T. C. , Neel K. L. , Lee K. H.
Published: January 2005

Previous studies have shown that peroxide caused electrophysiological damage. The present study investigates the action of agents that interfere with a free radical process in an effort to define the mechanism of peroxide damage. Deferoxamine chelates iron, making it unavailable for the Fenton reaction and thereby preventing the formation of hydroxyl free radicals from peroxide. Dimethylsulfoxide (DMSO) scavenges hydroxyl free radicals. Trolox‐C, a water soluble Vitamin E analog, is an antioxidant that can scavenge peroxy radicals. Slices of hippocampus were removed from brains of euthanized guinea pigs. Electrical stimulation of an orthodromic pathway to CA1 region evoked a synaptic response and a population spike. Input‐output curves were generated to evaluate the protection by deferoxamine, Trolox‐C, and DMSO on the synaptic damage and impaired spike generation caused by peroxide. Lipid peroxidation was measured by the thiobarbituric acid test. Peroxide was found to increase lipid peroxidation. Deferoxamine and Trolox‐C protected against the peroxide‐induced synaptic damage, impaired spike generation, and lipid peroxidation. DMSO was ineffective synaptically but reduced peroxide damage to spike generating mechanisms and further lipid peroxidation. The data support the hypothesis that peroxide causes damage through a free radical mechanism.
Transforming growth factor‐β and γ‐interferon have dual effects on growth of peripheral glia
Authors: Eccleston P. A. , Jessen K. R. , Mirsky R.
Published: January 2005

The influence of transforming growth factor‐β (TGF‐β) and γ‐interferon on DNA synthesis in Schwann cells and enteric glia in culture has been studied. TGF‐β stimulated the DNA synthesis of short‐term (less than 2 weeks in culture) Schwann cells, whereas γ‐interferon was ineffective. The stimulatory effect of TGF‐β was additive to the stimulation of DNA synthesis due to axonal membrane fragments. In contrast to their effect on short‐term Schwann cells, both TGF‐β and γ‐interferon inhibited DNA synthesis in enteric glial cells and in long‐term (over 3 months in culture) Schwann cells. When short‐term Schwann cells were stimulated to divide by axolemma or glial growth factor, γ‐interferon did not inhibit this enhanced DNA synthesis although it suppressed DNA synthesis induced by cAMP analogues. These results raise the possibility that TGF‐β and γ‐interferon might have a role in controlling glial proliferation during development and/or regeneration of the peripheral nervous system.
Simultaneous up‐regulation of neurofilament proteins during the postnatal development of the rat nervous system
Authors: Schlaepfer W. W. , Bruce J.
Published: January 2005

The expression of neurofilament (NF) proteins was examined during postnatal development of the rat nervous system in order to elucidate the nature of NF expression during the period of transition from the embryonic (immature) to the adult (mature) stages of NF expression. mRNAs to the light (NF‐L), mid‐sized (NF‐M), and heavy (NF‐H) NF proteins were compared by Northern blots and by in situ hybridizations, in NF‐rich (i.e., DRG, spinal cord and brainstem) and in NF‐poor (i.e., cerebellum and cerebral cortex) regions of the developing rat nervous system. NF proteins were analyzed by gel electrophoresis and by immunoblots. In each tissue, the expression of NF‐H was delayed compared to that of NF‐L and NF‐M, as previously reported. The present study now shows that the dealayed compared to that of NF‐L and NF‐M, as previously reported. The present study now shows that the delayed expression of NF‐H is accompanied by parallel up‐regulations in the expressions of NF‐L and NF‐M, both at the levels of mRNA and protein. Similar rates of increase of all three NF mRNAs occur between postnatal days 5 (P5) and 24 (P24) in rat spinal cord and DRG. Furthermore, the postnatal up‐regulation of NF expression is characterized by a progressive accumulation of all three NF proteins in the tissues. The findings indicate that the adult pattern of NF expression (with high levels of expression of all three NF proteins) becomes established during the postnatal period of development, raising questions as to the nature of factors that coregulate the expression of NF subunits.
Activation of arginine metabolism to glutamate in rat brain synaptosomes in thioacetamide‐induced hepatic encephalopathy: An adaptative response?
Authors: Albrecht J. , Hilgier W. , Rafalowska U.
Published: January 2005

Crude (P2) synaptosomes derived from rats with acute hepatic encephalopathy (HE) induced with thioacetamide showed a slightly increased uptake of radiolabeled arginine (ARG) and a 2.5‐fold enhanced conversion of newly taken‐up ARG to both glutamate (GLU) and γ‐aminobutyric acid (GABA) as compared with control synaptosomes with a high potassium medium decreased their radioactive GLU and GABA content without affecting the content of the precursor ARG. This result, which was identical with control or HE preparations, appears to indicate that ARG contributes at least, in part, to the synthesis of neurotransmitter GLU or GABA. As measured in purified synaptosomal preparations, HE increased by about 50% the activities of arginase and ornithine‐δ‐aminotransferase‐the two enzymes of the ARG to GLU shunt. It is postulated that increased conversion of ARG to GLU may compensate for excessive utilization of the latter amino acid as an ammonia trap during HE and, as such, may be considered as an adaptative response of the synaptic compartment to this pathological condition.
Regulation of apolipoprotein E gene expression after injury of the rat sciatic nerve
Authors: LeBlanc A. C. , Poduslo J. F.
Published: January 2005

The expression of apolipoprotein E (apo E) is dramatically increased following peripheral nerve injury. This increased expression has been postulated to be negatively influenced by unknown mechanisms during subsequent axonal regeneration (Muller et al.: Science 228:499–501, 1985). The present study investigates the role of the regenerating axon in regulating apo E gene expression in two experimental paradigms which permit or prevent axonal regeneration in the adult rat sciatic nerve—the crush or permanent transection injuries. The nerves in these two models undergo axonal degeneration, demyelination, and Schwann cell proliferation; however, subsequent axonal regeneration and remyelination occur only in the distal segment of the crush‐injured and not in the permanently transected nerve. The steady‐state levels of apo E mRNA in both models increase sharply between 1 and 4 days and reach a maximum level at 12–14 days, which did not change significantly between 14 and 35 days after injury. No significant difference is observed in the steady‐state levels of apo E mRNA between the crushed and permanently transected nerves as a function of time after injury. The steady‐state protein level of apo E in the endoneurial segments initially increases, peaks at 14–21 days, and then decreases between 35 and 60 days after injury in both models. In contrast, the rate of newly translated and secreted apo E significantly increases by fourfold (P < 0.005) between 35 and 60 days after permanent transection whereas it does not significantly differ at these times after crush injury. The increased rate of translation and secretion of apo E after transection compared to the constant rate observed after crush injury, together with the comparable steady‐state levels of apo E mRNA and protein in both models, suggests translational or post‐translational control, but not transcriptional and/or post‐transcriptional control, by the regenerating axons. Furthermore, the increasing rate of biosynthesis and secretion of apo E after permanent transection concomitant with the decreasing steady‐state levels of the protein suggests that apo E is either removed from the endoneurium or subsequently utilized or degraded by mechanisms that are independent from nerve regeneration.
Development of A1 adenosine receptors in the chick embryo retina
Authors: de Carvalho R. Paes
Published: January 2005

Adenosine inhibits cyclic AMP synthesis induced by dopamine in embryonic but not in post‐hatched chick retinas. N6‐Cyclohexyladenosine (CHA), which preferntially activates A1 receptors as well as 2‐chloroadenosine, inhibits cyclic AMP accumulation induced by dopamine in retinas from 10‐day‐old embryos (E10) with IC50's of 0.1 and 0.5 μM, respectively, but this effect is not detectable after hatching. In order to verify if this developmental change reflects variations in the number of affinity of A1 adenosine receptors, their development during chick retina ontogeny was studied. Binding studies using 3(H)CHA revealed the presence of A1 receptors at all stages of development examined, including the post‐hatched retina. The number of binding sites increased between E10 and E17, and then decreased in post‐hatched animals. In the latter, 3(H)CHA binding was to a single site with a Bmax of 128.6 ± 13.4 fmol/mg protein and a Kd of 2.1 + 0.2 nM. Various ligands showed similar hierachies of affinity for the A1 receptor in embryonic and post‐hatched retinas, namely, CHA > R‐N6‐phenylisopropyladenosine (1‐PIA)> 5′,‐N‐ethylcarbox‐amideadenosine (NECA) > isobuthylmethyl‐xanthine (IBMX). Given that CHA inhibited forskolin‐induced cyclic AMP production and Gpp(NH)p inhibited 3(H)CHA binding in both embryonic and posthatched retinas, it appears that receptor coupling to adenylate cyclase is present since early embryonic stages. The results suggest that the A1 receptors may have different functions in the embryonic as compared to the mature chick retina.