bluuebunny/crossref_metadata_2025 huggingface dataset:
Developmental expression of the gene encoding growth‐associated protein 43 (GAP43) in the brains of normal and aneuploid mice
Authors: Capone G. T. , Bendotti C. , Oster‐Granite M. L. , Coyle Joseph T.
Published: January 2005

The gene encoding growth‐associated protein 43 (Gap43), a neuronal phosphoprotein associated with axonal outgrowth and synaptic plasticity, is located on mouse chromosome 16 (MMU16). We examined the developmental expression of Gap43 in normal, trisomy 16 (Tsl6), and trisomy 19 (Tsl9) mouse brain using northern blot analysis and in situ hybridization as a first step toward understanding the neurobio‐logic consequences of increased gene dosage on brain development. Gap43 expression was detected by in situ hybridization throughout the mesencephalon, rhombencephalon, spinal cord, and first branchial arch in whole embryos as early as (Jay 10 of gestation (E10). By E15, Gap43 expression was localized to cells in the retina, the olfactory bulbs, and anterior olfactory structures, the cortical plate, the basal telen‐cephalon, diencephalon, midbrain, hindbrain, ana spinal cord. Northern blot analysis detected a threefold increase in Gap43mRNA levels in the brains of normal mice between E12–E18. At E15, Gap43 mRNA levels were increased 35–40% in Tsl6 mouse brain and decreased 10% in Tsl9 mouse brain, relative to euploid littermate controls. Using in situ hybridization we found that overexpression of Gap43 occurred in the diencephalon, medial and lateral basal telencephalon, and cortical plate region in Tsl6 mice relative to littermate controls. Thus, the degree of overexpression of Gap43mRNA in Ts16 mice is consistent with that expected from gene dosage effects.
Temporal pattern of innervation in the developing mouse inner ear: An immunocytochemical study of a 66‐kD subunit of mammalian neurofilaments
Authors: Galinovic‐Schwartz V. , Peng D. , Chiu F. C. , Van De Water T. R.
Published: January 2005

We have examined the expression of a 66‐kD neurofilament protein (NF‐66) in the developing inner ear. Mouse embryos, fetuses, and neonates were fixed in Methacarn, embedded in paraffin, and sectioned. A polyclonal antiserum raised specifically to NF‐66 and unreactive to NF‐L, ‐M, ‐H, and peripherin was used for immunocytochemical staining. NF‐66 immunostaining was first detectable in the rhombencephalon at embryonic day (E) 9.5. Immunoreactivity was first detected in the statoacoustic ganglion (SAG) early on E10.5. By late E10.5, the first SAG axons were detectable within the intraepithelial spaces of the otocyst. At E12, NF‐66 positivity was detectable in neurites that projected into areas of presumptive vestibular sensory epithelium. Neurites projecting into the presumptive acoustic sensory epithelium were negative. However, at E13, the projections from both the vestibular and the acoustic ganglion (i.e, cochlear duct) were both NF‐66 positive. In the cell bodies, NF‐66 expression appeared earlier in the vestibular than in the auditory neurons. By E16, neuronal somas in both ganglia were NF‐66 positive.
Studies of glial lineage and proliferation in vitro using an early marker for committed oligodendrocytes
Authors: Knapp P. E.
Published: January 2005

The potential of immature glial cells to differentiate into astrocytes (ASs) or oligodendrocytes (OLs) has been examined using a monoclonal antibody (007) that is specific for OLs in vivo. Cells were dissociated from 2‐day postnatal mouse cortex and labeled with the 007 antibody 2 hr after plating. The cells which were labeled during this single, brief exposure to the antibody retained the antibody on their surfaces over the course of the experiments. Cells were double stained at various timepoints for residual 007 antibody and either galactocerebroside (GC) or glial fibrillary acidic protein (GFAP). Shortly after plating, most 007 + cells were GC − and none expressed GFAP. These cells were round, although some had begun to extend very short processes. After 96 hr, >95% of cells with residual 007 on their surfaces also expressed GC. By this time, all the 007 + cells had several processes of varying lengths extending from their cell bodies. Cells expressing both 007 and GFAP were never seen. The 007 + /GC + OLs were not induced to differentiate from 007 + bipotential progenitors since they were grown in fetal calf serum. These results show that under our culture conditions the 007 antibody is OL specific. Immunostaining for bromodeoxyuridine, a marker for dividing cells, revealed that some 007 + cells were proliferating. The majority of these proliferating cells had already extended three or more processes. We therefore conclude that immature, process‐bearing cells can be committed to the OL lineage at times before they express detectable amounts of GC. Since these young 007 + OLs are actively proliferating, committed cells can serve as an important source of new OLs.
Regulation of neuronal differentiation in neuron‐enriched primary cultures from embryonic rat cerebra by platelet activating factor and the structurally related glycerol ether lipid, dodecylglycerol
Authors: Ved H. S. , Gustow E. , Pieringer R. A.
Published: January 2005

Primary cultures enriched in neurons dissociated from embryonic rat cerebra were used to demonstrate that platelet activating factor and the structurally related ether glycerolipid, dodecylglycerol, are readily taken up in small amounts by neurons and that they stimulate the differentiation of neurons. The stimulation of neuronal differentiation was observed as a precocious development of axon‐like extensions which correlated with a concentration — dependent increase in neuronal‐specific enzyme activities. This stimulation of morphological and neurochemical factors by either platelet activating factor or dodecylglycerol was almost completely abolished by triazolam, a known inhibitor of platelet activating factor function. Neither platelet activating factor nor dodecylglycerol at the concentrations used to achieve stimulation of neuronal differentiation compromised the plasma membrane, as indicated by the lack of leakage of cytoplasmic lactic acid dehydrogenase.
Conditioned media derived from glial cell lines promote survival and differentiation of dopaminergic neurons in vitro: Role of mesencephalic glia
Authors: Engele J. , Schubert D. , Bohn M. C.
Published: January 2005

Neuronal differentiation is influenced by extracellular factors; however, only a few such factors have been identified for central neurons. To address this issue, we have screened media conditioned (CM) by several glial cell lines for neurotrophic effects on dopaminergic neurons in dissociated cell cultures of the E14.5 rat mesencephalon grown in serum‐free conditions. To establish culture conditions under which dopaminergic cell survival depends on the exogenous support from neurotrophic factors, cell suspensions were seeded at varying densities and the number of tyrosine hydroxylase‐immunoreactive (TH‐IR) neurons was determined. This number was maximal at plating densities greater than 175,000 cells/cm2 and was 10‐fold lower at the plating density of 80,000 cells/cm2. Cell density had only a minimal effect on [3H]dopamine uptake per TH‐IR neuron. Treatment of cultures plated at 80,000 cells/cm2 with CM derived from the glial cell line, B49, the neural retina glial cell line, R33, and the Schwannoma cell line JS1, increased the number of surviving TH‐IR neurons 160–330%. These effects were dose dependent and heat sensitive. All CM stimulated neurite elongation of TH‐IR neurons, while only the B49‐CM increased [3H]dopamine uptake. The neurotrophic effects of these media were not confined to dopaminergic neurons but increased overall neuronal density in culture by 50–100%. Moreover, all three CM were mitogenic for mesencephalic glia as demonstrated by glial fibrillary acidic protein (GFAP)‐immunocytochemistry in combination with [3H]thymidine‐autoradiography. By contrast, medium conditioned by the pheochromocytoma cell line, PC 12, did not increase the number of astrocytes or promote the survival of dopaminergic neurons. Inhibition of glial proliferation reduced the neurotrophic effects of the B49‐, R33‐, and JS1‐CM by 40–80%.

These observations suggest that the glial cell lines B49, R33, and JS1 secrete factors that promote the survival of dopaminergic neurons and induce proliferation of glial precursors. The partial decrease of the survival‐promoting effects of these CM on dopaminergic neurons in glial‐free mesencephalic cultures further suggests that the observed neurotrophic effects result from the combined action of cell line‐derived substances directly on neurons and indirectly via effects on mesencephalic astrocytes or astrocyte precursors.
Internalization of the inhibitor of the N‐acetylgalactosaminyltransferase by chicken embryonic retina cells: Reversibility of the inhibitor effects
Authors: Quiroga S. , Panzetta P. , Caputto R.
Published: January 2005

Retina cells from 6‐day‐old chicken embryos were cultured in the resence of an 125I‐labeled protein inhibitor of the UDP‐N‐acetylgalactosamine:GM3,N‐acetylactosaminyltransferase. The cells were labeled and did not lose the incorporated radioactivity when treated with 0.125% trypsin or 1 M NaCl at 37°C for 1 hr, indicating that the iodinated inhibitor was inside the cells. Immunostaining procedures using an anti‐inhibitor was antibody were applied to the cells cultured in the presence of the inhibitor after permeabilization of the cells. The inhibitor was found inside the round cells virtually devoid of neurites, but not in flat glial‐like cell or in process‐bearing neural cells. Also found was an apparent self‐recovery effect of the cells for both the anti‐neuritogenic effect and the modification of the pattern of labeled gangliosides produced by the inhibitor when the agent was withdrawn from the culture medium after the initial period of 20 hr. This recovery was clearly observed 72 hr after the removal of the inhibitor.
Host response during successful engraftment of fetal xenogenic astrocytes: Predominance of microglia and macrophages
Authors: Booss J. , Baumann N. , Collins P. , Jacque C.
Published: January 2005

Grafting of fetal rabbit brain fragments into the brains of newborn mice results in the successful establishment and migration of xenogenic astrocytes in the majority of recipients. This can be demonstrated by the use of Tp‐GFAP1 monoclonal antibody which binds with rabbit, but not with murine glial fibrillary acidic protein. In the first phase, donor astrocytes are found in more than 80% of recipients 3 and 4 weeks after grafting. In the second phase, there is a decline and disappearance of donor astrocytes by the tenth week. We have recently demonstrated that the decline and disappearance of donor astrocytes was coincident with infiltration of T cells into the brain, compatible with T‐cell‐mediated graft rejection. In the present studies, we wished to characterize the types of host cells responding during the period of graft success, in the first 4 weeks after transplantation. It was found that responses by microglia, macrophages, and astrocytes occurred promptly and were sustained throughout this period. Host responses occurred at the graft site and at sites of migration. Examination of sham transplanted control mice revealed responses by the same types of cells. No expression of donor Ia antigen was observed, and the expression of Ia antigen by the host was variable and of low magnitude. T cells were rarely present in transplanted brains during this period.

Taken together with previous findings, the present studies demonstrate a clear difference in the host response in the brain at the time when xenogenic astrocytes migrate and survive compared to the period when they disappear. The biphasic nature of the model should facilitate studies to examine mechanisms of brain graft rejection as well as the design of protocols to prevent rejection.
Isoprenoid modification permits 2′,3′‐cyclic nucleotide 3′‐phosphodiesterase to bind to membranes
Authors: Braun P. E. , De Angelis D. , Shtybel W. W. , Bernier L.
Published: January 2005

The myelination‐related enzyme 2′,3′‐cyclic nucleotide 3′‐phosphodiesterase (CNP), a relatively abundant protein in the CNS possesses the C‐terminal isoprenylation consensus domain found in a small family that includes the ras oncoproteins and their relatives, some G‐proteins, and nuclear lamins. We found that CNP, like these other proteins, is modified post‐translationally by an isoprenoid derived from mevalonic acid. It appears that only the smaller of the two CNP isoforms (CNP1) is isoprenylated, but similar modification of CNP2 cannot be excluded. Inhibition of isoprenoid synthesis by Lovastatin blocks the binding of newly synthesized CNP to cell membranes; binding is restored upon addition of mevalonate to the culture medium. This shows that isoprenylation is permissive for the well‐known avid association of CNP with membranes.
Effect of acetyl‐L‐carnitine on the dopaminergic system in aging brain
Authors: Sershen H. , Harsing L. G. , Banay‐Schwartz M. , Hashim A. , Ramacci M. T. , Lajtha A.
Published: January 2005

We studied the effect of acetyl‐L‐carnitine (ALCAR) on dopamine release and the effect of long‐term acetyl‐L‐carnitine treatment on age‐related changes in striatal dopamine receptors and brain amino acid levels.

In striatal tissue that had been incubated with [3H]dopamine, acetyl‐L‐carnitine increased the release of [3H]dopamine evoked by electrical stimulation. In striatal tissue from aged mice administered acetyl‐L‐carnitine for 3 months, the release of [3H]dopamine evoked by electrical stimulation was higher than that of its aged control; the release after a second stimulation was similar in the two groups.

There was a significant decline in the number of D1 striatal dopamine receptors with age. The Bmax was 51% lower in 1.5‐year‐old mice than in 4‐month‐old animals. Administration of acetyl‐L‐carnitine for 3 months diminished the reduction in the binding of [3H]SCH‐23390. [3H]Spiperonebinding to D2 receptors was not decreased with age and was not affected by acetyl‐L‐carnitine treatment.

Age‐related decreases in levels of several amino acids were observed in several brain regions. Acetyl‐L‐carnitine lessened the reduction in the level of taurine only in the striatum.

The findings confirm the multiple effects of acetyl‐L‐carnitine in brain, and suggest that its administration can have a positive effect on age‐related changes in the dopaminergic system.
Evidence that protein constituents of postsynaptic membrane specializations are locally synthesized: Time course of appearance of recently synthesized proteins in synaptic junctions
Authors: Steward O. , Pollack A. , Rao A.
Published: January 2005

Previous studies have led to the hypothesis that some protein constituents of postsynaptic membrane specializations are locally synthesized near postsynaptic sites. The present study focuses on one prediction of this hypothesis, specifically, that if some proteins of the postsynaptic membrane specialization are locally synthesized, then the delay between synthesis and assembly into synaptic junctional membrane could be short. We evaluate the time course of appearance of recently synthesized protein in synaptic junctions by pulse‐labeling hippocampal slices maintained in vitro with radiolabeled protein precursors, and then isolating subcellular fractions enriched in synaptic plasma membranes (SPM) and synaptic junctional complexes (SJC). We report that there is no evidence of a delay in the appearance of recently synthesized proteins in SPM and SJC fractions. Labeled proteins could be detected as early as 15 min after the initiation of the pulse‐labeling period, and the extent of labeling increased monotonically thereafter. The labeling could not be accounted for by contamination of synaptic membrane fractions with other membranes, because the relative specific activity of the SPM and SJC fractions was the same or higher than that of the less pure fractions from which these synaptic fractions were derived. One‐dimensional PAGE‐fluorography was used to provide an initial characterization of which proteins were labeled in SJC fractions. We found that the most prominent labeled bands were at apparent molecular weights of approximately 43–44, 55–56, and 60 kd, with more lightly labeled bands at about 38 and 116 kd. In some preparations, there was a labeled doublet at about 36–38 kd. There were also other lightly labeled bands at other molecular weights. These bands were much less heavily labeled than the bands at 43–44, 55–56, and 60 kd, however. There was little labeling in the molecular weight range of the “major psd protein” (the alpha subunit of CAM‐kinase), although there was diffuse labeling throughout the 45–52 kd region. These results are consistent with the hypothesis that some of the protein constituents of the postsynaptic junctional complex are synthesized by polyribosomes which are selectively localized beneath synaptic junctions.
Association between vascular basement membrane components and the lesions of Alzheimer's disease
Authors: Perlmutter L. S. , Barrón E. , Saperia D. , Chui H. C.
Published: January 2005

A relationship between the microvasculature and Alzheimer senile plaques has been suggested by several lines of evidence. Besides close anatomic and biochemical relationships, both extrinsic (fibronectin) and intrinsic [heparan sulfate proteoglycan (HSPG)] components of the vascular basement membrane (VBM) have been colocalized with amyloid plaques. The present study was designed to examine the association between three intrinsic components of the VBM [HSPG, collagen type IV (CIV), and laminin] and the histopathologic lesions of Alzheimer's disease (AD).

Six cases with neuropathologically confirmed AD were immunocytochemically labeled for the presence of HSPG, CIV, laminin, or tau‐2 (a marker for degenerating neurites) and examined at the light and electron microscopic levels. For light microscopic analyses, sections were counterstained with a fluorescent marker for amyloid. The present study illustrates an involvement of VBM components in the lesions associated with AD. First, we replicate our previous finding that HSPG antibodies immunolabel a subset of neurons; ultrastructural analyses indicate that at least some of these are actually extracellular neurofibrillary tangles. Second, we report that CIV and laminin immunoreaction product was not associated with neurons but did label several perivascular cells with the morphologic characteristics of microglia. Finally, we demonstrate that all three intrinsic VBM components, CIV and laminin as well as HSPG, are localized to senile plaques. Both light and electron microscopic studies indicate that the VBM components are associated with amyloid rather than degenerating neurites. These findings suggest that the VBM or its components may play a role in the AD pathogenetic cascade.
Immunolocalization of Alzheimer β‐amyloid peptide precursor to cellular membranes in baculovirus expression system
Authors: Currie J. R. , Ramakrishna N. , Burrage T. G. , Hwang M.‐C. , Potempska A. , Miller D. L. , Mehta P. D. , Kim K. S. , Wisniewski H. M.
Published: January 2005

One characteristic of Alzheimer's disease (Aβ disease) is the accumulation of amyloid deposits within the extracellular space of the brain and meninges. A 40 amino acid peptide called β‐peptide or A4 protein is the subunit of the amyloid fibrils found in these deposits. The sequence of β‐peptide is contained within those of a family of larger proteins called the Alzheimer β‐amyloid peptide precursor (APP). These APPs contain, in addition to a signal sequence, a hydrophobic sequence that is believed to span cell membranes. Although biochemical studies indicate that some APPs have properties of integral membrane proteins, morphological confirmation of this has not been reported. We recently described an expression system in which human APP751 cDNA was placed under the transcriptional regulation of the polyhedrin gene promoter in the baculovirus Autographica californica infecting a Spodoptera frugiperda cell line (Ramakrishna et al., Biochem Biophys Res Commun 174:983–989, 1991). As part of a larger biochemical and molecular biological study of APP, we have carried out an immunocytochemical study using antibodies directed against several epitopes within APP to reveal, at both the light and the electron microscopic levels, the cellular localization of APP in the baculovirus expression system. These studies demonstrate that APP751 is abundantly synthesized and inserted into certain of the membrane compartments of the cell. As early as 24 hr postinfection, APP751 is found associated with all membrane compartments excepting mitochondrial membranes. The patterns of immunolabeling are consistent with our biochemical findings that the protein is processed in these cells so as to release the extracellular domain and to retain a transmembrane and intracellular segment. These data provide the first morphological demonstration of the membrane location of APP751 its posttranslational processing to a secreted fragment, and its exclusion from the mitochondrial membranes. This system is especially valuable for identifying conditions under which antibodies raised against APP or appropriate synthetic peptides will react with native APP.
Structure, expression, and function of, Ng‐CAM a member of the immunoglobulin superfamily involved in neuron‐neuron and neuron‐glia adhesion
Authors: Grumet M.
Published: January 2005

The neuron‐glia cell adhesion molecule (Ng‐CAM) mediates neuron‐neuron adhesion by a homophilic mechanism and neuron‐astrocyte adhesion by a heterophilic mechanism. The protein is expressed on neurons and Schwann cells but not on astrocytes. It is most prevalent during development on cell bodies of migrating neurons and on axons during formation of nerves. Ng‐CAM expression is greatly increased following nerve injury. Anti‐Ng‐CAM antibodies inhibited migration of granule cells along Bergmann glia in cerebellar explants and fasciculation of neurites in outgrowths from explants of dorsal root ganglia. The combined results indicate that Ng‐CAM on neurons binds to Ng‐CAM on adjacent neurons and to as yet unidentified ligands on astrocytes.

Ng‐CAM is synthesized in chicken neurons from a 6 kb mRNA as Mr ∼200,000 forms which are cleaved to yield two components of Mr 135,000 and 80,000. It is glycosylated and can be phosphorylated. Amino acid sequence analysis indicates that it contains six immunoglobulin domains, five fibronec‐tin type III repeats, a transmembrane domain and a cytoplasmic region. Structural analyses indicate that Ng‐CAM is most closely related to the mammalian glycoprotein L1 but significant differences between them strongly suggest that they are not equivalent molecules. The recent identification of another structurally related molecule in the chicken called Nr‐CAM underscores the notion that these molecules are members of a subfamily of neural cell adhesion molecules within the immunoglobulin superfamily that have related or complementary functions in the nervous system.
Glycerophosphorylcholine phosphocholine phosphodiesterase activity in cultured oligodendrocytes, astrocytes, and central nervous tissue of dysmyelinating rodent mutants
Authors: Yuan J. , McCartney D. G. , Monge M. , de Los Monteros A. Espinosa , Zalc B. , de Vellis J. , Kanfer J. N.
Published: January 2005

The levels of GPC phosphocholine phosphodiesterase, pNP phosphocholine phosphodiesterase, CNPase, and UDP galactose: ceramide galactosyltransferase activities were estimated with pure cultures of oligodendrocytes and astrocytes; mixed primary glial cells cultures; C‐6 cells; and CNS tissue of the dysmyelinating md rat, the jimpy mouse, and the quaking mouse. The highest activity of GPC and pNP phosphocholine phosphodiesterases as with CNPase and C gal T was found in the pure cultured oligodendrocytes. C‐6 cells had very low or undetectable activities for these two phosphodiesterases but possessed very high CNPase activity. The activity of GPC phosphocholine phosphodiesterase was significantly decreased in the CNS tissue of the md rat and the jimpy and the quaking mouse. Similar reductions were observed for the pNP phosphocholine phosphodiesterase, CNPase, and C gal T activities. The selective cellular enrichment in oligodendrocytes of the GPC phosphocholine phosphodiesterase activity and decreases of its activity in three dysmyelinating mutants in the same ratio as for CNPase and C gal T suggest that GPC phosphocholine phosphodiesterase is a myelin marker enzyme and it may reflect the quantity of myelin and oligodendrocyte present.
Interaction between oligodendroglia and immune cells: Mitogenic effect of an oligodendrocyte precursor cell line on syngeneic T lymphocytes
Authors: Yamamura T. , Sun D. , Aloisi F. , Klinkert W. E. F. , Wekerle Hartmut
Published: January 2005

We analyzed cellular interactions between T lymphocytes and a recently established immortal glial line, L3 that retains several properties of immature oligo‐dendrocytes (Aloisi et al., J Neurosci Res 27:16–24, 1990). L3 oligodendrocytes (L3‐OL) cannot be induced to express class II antigens, nor do they specifically present antigen to syngeneic specific T lymphocyte. However, L3‐OL strongly enhance the proliferation of freshly activated, interleukin‐2(IL‐2)‐dependent T‐line lymphocytes and concanavalin A (ConA)‐activated lymphoblasts, irrespective of their antigen specificity or surface phenotype (CD4+ or CD8+). Resting and some activated T cells were susceptible to the mitogenic effect of L3‐OL only in the presence of exogenous IL‐2, not of other cytokines. The mitogenic effect of L3‐OL did not depend on cell viability. It was observed in paraformaldehyde‐fixed L3‐OL cells and in membrane preparations, but not in culture supernatant. Neither intact L3‐OL cells nor membrane preparations had direct IL‐2 activity. The conclusion that the mitogenic effect of L3‐OL cells is exerted by membrane structures acting as a costimulatory factor(s) of IL‐2 is supported by the finding that it is largely blocked by a monoclonal anti‐IL‐2 receptor antibody. The effect is distinct from membrane‐bound IL‐1, membrane‐bound tumor necrosis factor‐α (TNF‐α), IL‐3, or IL‐6 and cannot be reconstituted by these cytokines. © 1992 Wiley‐Liss, Inc.
Microtubule‐associated protein tau is required for axonal neurite elaboration by neuroblastoma cells
Authors: Shea T. B. , Beermann M. L. , Nixon R. A. , Fischer I.
Published: January 2005

NB2a/d1 neuroblastoma cells constitutively express multiple isoforms of the microtubule‐associated protein tau and incorporate this protein into the axonal neurites elaborated during serum deprivation. To examine whether or not tau played an essential role in axonal outgrowth, cells cultured in serum‐free medium were treated at 24 h intervals with antisense‐ and sense‐oriented cDNA oligonucleotides (25 or 36 mers that span or are upstream of tau initiation codon) and were simultaneously serum deprived. Oligonucleotide uptake was confirmed by determination of intracellular levels of radiolabeled oligonucleotides. Treatment for 48 h with tau antisense oligonucleotides reversibly inhibited the expression of tau and the number of neurite‐bearing cells compared with treatment with sense oligonucleotides. By contrast, tubulin expression was not affected. When cells were treated with antisense oligonucleotide simultaneously with serum deprivation, the initial outgrowth of neurites was unaffected, but continued neurite elongation was prevented. By contrast, neurite outgrowth at 4 h was inhibited when cells were pretreated with tau antisense 24 h before serum deprivation. Furthermore, intracellular delivery of antitau antiserum prevented neurite outgrowth and, in cells that had previously been deprived of serum for 24 h, induced retraction of existing neurites. These findings indicate that both the initiation and the continued outgrowth of neurites are dependent on tau and that pre‐existing cytoplasmic pools of tau can mediate initial neuritogenesis. © 1992 Wiley‐Liss, Inc.
The morphological localization and biochemical characterization of a synapsin I‐like antigen in the nervous system of Aplysia californica
Authors: Bongiovi M. E. , Ambron R. T. , Silverman A.‐J.
Published: January 2005

Synapsins are a well‐characterized class of phosphoproteins found at synapses in the mammalian nervous system. One member of this family, synapsin I, has been extensively studied and shown to associate in a phosphorylation‐dependent manner with both small synaptic vesicles and cytoskeletal elements. Though the characteristics of synapsin I suggest an important function in synaptic transmission, its definitive role is still in question. In an effort to find a model system in which to test directly the function of synapsin I, we have looked in the nervous system of the marine mollusc Aplysia californica for synapsin I‐like antigens (SILA).

Light microscope immunocytochemical studies using polyclonal and monoclonal antibodies to bovine brain synapsin I demonstrate Aplysia SILA in neuronal somata, in the neuropil, and at some identified synapses. Though SILA were exclusively associated with neuronal structures in Aplysia, the pattern of staining suggested that they are not present at all synaptic terminals. This interpretation was corroborated by ultrastructural studies in which SILA were present at some synaptic terminals but absent, or in low abundance, in adjacent terminals. In axons, SILA were associated with vesicles of 120–150 nm diameter, as well as with filamentous structures. Biochemical studies identified small amounts of SILA of 40 and 50 kD molecular weight that are recognized by several antibodies to mammalian synapsin I, and are acid extractable, collagenase‐sensitive phosphoproteins; these are criteria used to define synapsin I homologues in other species. Our studies indicate that SILA are present in neurons in Aplysia californica but suggested that they represent only a small percentage of the total protein within the nervous system. © 1992 Wiley‐Liss, Inc.
Nerve growth factor receptor gene expression in human peripheral blood lymphocytes in aging
Authors: Kittur Smita D. , Song L. , Endo H. , Adler W. H.
Published: January 2005

Nerve growth factor (NGF) has a modulating effect on immune function, which may occur as a consequence of binding to the NGF receptor (NGF‐R). To determine if mRNA for the gene coding for p75NGFR (low affinity NGF‐R) is present in lymphocytes, Northern blot analysis of mRNA from human peripheral blood lymphocytes (PBL) and purified T lymphocytes was initiated using cDNA probe for human p75NGFR. p75NGFR mRNA was present in PBL and T lymphocytes, and the mRNA in response to phytohemagglutinin stimulation showed maximum levels at 14 hr of stimulation. p75NGFR mRNA content when analyzed in PBL and T cells from volunteers of various ages showed that p75NGFR mRNA expression does not change with the age of the cell donor. © 1992 Wiley‐Liss, Inc.
Differential expression of the presynaptic protein SNAP‐25 in mammalian retina
Authors: Catsicas S. , Catsicas M. , Keyser K. T. , Karten H. J. , Wilson M. C. , Milner R. J.
Published: January 2005

We have studied the expression of the nerve terminal protein synaptosomal associated protein 25 (SNAP‐25) in the retina of adult rat, mouse, and monkey, as well as in the developing mouse retina. To evaluate SNAP‐25 expression, its distribution was compared to those of the synaptic vesicle‐associated proteins synapsin I and synaptophysin. In situ hybridization in adult rat retinas suggested that SNAP‐25 mRNA is mainly expressed by ganglion, amacrine, and horizontal cells, but not by photoreceptors and bipolar cells. In all species, the SNAP‐25 polypeptide was most abundant in the inner part of the inner and outer plexiform layers and was also found in the ganglion cell axons. In adult retina, synapsin I and synaptophysin were also mainly localized in synaptic fields and processes but all three proteins showed a distinct pattern of distribution. Finally, in mouse retina, the three proteins were first detectable at embryonic day 16 and subsequently showed developmentally regulated changes in their cellular localization. These results suggest that SNAP‐25 is predominantly expressed in specific subtypes of conventional synapses, but not ribbon synapses, and that it may also be involved in the physiology of nonvesicular terminals of horizontal cells. Our study also suggests that combinatorial expression of different components of the presynaptic specialization may contribute to synaptic functional diversity. © 1992 Wiley‐Liss, Inc.
Cold‐induced alterations in the binding of adrenomedullary nuclear proteins to the promoter region of the tyrosine hydroxylase gene
Authors: Miner L. L. , Pandalai S. P. , Weisberg E. P. , Sell S. L. , Kovacs D. M. , Kaplan B. B.
Published: January 2005

It is well documented that cold stress induces a rapid trans‐synaptically mediated increase in the relative abundance of rat adrenomedullary tyrosine hydroxylase (TH) mRNA. To investigate the transcriptional mechanisms regulating the cold stress response, we have employed a gel mobility shift assay, using DNA fragments prepared from the proximal 5′ flanking region of the bovine TH gene as a heterologous molecular probe. In pilot studies, this region of the bovine TH promoter (nucleotides −246 to +21) was fused to the bacterial reporter gene, chloramphenicol acetyltransferase, and the chimeric construct transfected into human neuroblastoma SK‐N‐BE(2)‐C, hepatoma HepG2, and rat pheochromocytoma PC‐12 cells. Results of this analysis indicate that the proximal 5′ flanking region of the bovine TH gene contains sufficient information to drive transient reporter gene expression in both human and rat catecholaminergic clonal cell lines. The findings derived from the gel mobility shift studies demonstrate that cold exposure causes rapid and selective alterations in the binding of adrenomedullary nuclear proteins to the proximal 5′ flanking region of the TH gene. The most striking cold stress‐induced alteration in DNA/nucleoprotein binding occurs in a region of the TH promoter (nucleotides −246 to −189) which contains an element bearing marked sequence similarity to an AP1 binding site and is highly conserved among animal species. This alteration occurs within 1 hr of cold exposure and persists for up to 48 hr after the onset of stress. The results of adrenal denervation experiments indicate that the cold‐induced change in DNA/nucleoprotein binding is neurally mediated, requiring intact sympathetic innervation of the gland. Formation of the cold‐induced DNA/nucleoprotein complex was inhibited by competition with a synthetic oligodeoxyribonucleotide containing a consensus sequence for the AP1 binding site. Pretreatment of adrenomedullary nuclear extracts with antibodies to either c‐Fos or c‐Jun also inhibited the cold‐induced alteration in DNA/nucleoprotein binding. Heat denaturation of the anti‐Fos or anti‐Jun antibodies, or preadsorption of the antibodies with their cognate antigenic peptides eliminated the inhibitory effect. Taken together, these findings suggest that the cold‐induced increases in adrenomedullary TH gene expression are mediated, at least in part, through the interaction of a phorbol ester‐responsive element and proto‐oncogene transcription factors. © 1992 Wiley‐Liss, Inc.
Nitric oxide synthase is critical in mediating basal forebrain regulation of cortical cerebral circulation
Authors: Raszkiewicz J. L. , Linville D. G. , Kerwin J. F. , Wagenaar F. , Arneric S. P.
Published: January 2005

This study sought to determine whether the activity of nitric oxide synthase (NOS) is an important physiological link required to mediate increases in cortical cerebral blood flow (CBF) elicited by electrical microstimulation of the basal forebrain (BF). Changes in cortical CBF were assessed in urethane anesthetized rats using laser‐Doppler flowmetry. Microstimulation of the BF elicited stimulus‐locked increases in CBF that were dependent on frequency and current intensity (up to 280% of control at 50 Hz). Infusion of the potent NOS inhibitor NG‐nitro‐L‐arginine (L‐NNA) resulted in significant dose‐related reductions in the BF‐elicited response at 50 Hz (3.75–60 mg/kg, i.v.), significant elevation in resting mean arterial pressure (MAP) from 106 to 160 mmHg, and modest 21% reductions in resting CBF. The stereoisomer NG‐nitro‐D‐arginine (D‐NNA) was without any effect on CBF, although at higher concentrations MAP was elevated to levels comparable to those obtained with L‐NNA. Infusion of arginase was also without effect on resting or BF‐elicited CBF responses. In contrast, L‐arginine (100–400 mg/kg, i.v.) significantly potentiated the BF‐elicited response up to an additional 38%, without affecting resting CBF or MAP. This study suggests that NO, or a related nitroso precursor formed by NOS, has a critical role in mediating regulation of cortical CBF by BF neurons. © 1992 Wiley‐Liss, Inc.
Neurofilament and tubulin mRNA expression in schwann cells
Authors: Roberson M. D. , Toews A. D. , Goodrum J. F. , Morell P.
Published: January 2005

Feeding of elemental tellurium to weanling rats blocks synthesis of cholesterol (a major component of myelin), and causes demyelination of the sciatic nerve. Expression of mRNA for myelin‐specific genes in Schwann cells is downregulated. We now demonstrate specificity for Schwann cell injury in that expression of mRNAs for neurofilament subunits and for class II β‐tubulin (parameters sensitive to axonal injury) is unaltered in neurons of the dorsal root ganglia. An unexpected result was that in tellurium‐treated rats there was marked upregulation of expression of mRNAs coding for the light and medium neurofilament subunits (“neuron‐specific” proteins) as well as that for class II β‐tubulin (the major neuronal β‐tubulin isotype) in Schwann cells. Expression of these “neuronal” mRNA species was also detected in distal stumps of transected nerves at times when Schwann cells were undergoing dedifferentiation. © 1992 Wiley‐Liss, Inc.
S‐100β and insulin‐like growth factor‐II differentially regulate growth of developing serotonin and dopamine neurons in vitro
Authors: Liu J. P. , Lauder J. M.
Published: January 2005

To study the phenotypic specificity of S‐100β and insulin‐like growth factor II (IGF‐II) for developing monoamine neurons, serotonin (5‐HT) neurons from the embryonic day 14 (E14) rostral raphe or dopamine (TH) neurons from the substantia nigra/ventral tegmental area were cultured for 3 days in vitro (3 DIV) in the presence of these factors. Neuronotrophic effects were analyzed by computer‐assisted morphometry of 5‐HT and TH‐immunoreactive neurons. S‐100β significantly increased several parameters of neurite outgrowth by 5‐HT neurons but inhibited the spatial extent (field area) of TH neurites. IGF‐II promoted growth of cell bodies of both phenotypes, but only stimulated neurite outgrowth by TH neurons. S‐100β and IGF‐II differentially affected the number of GFAP immunoreactive cells from raphe and substantia nigra, but these effects did not correlate with the specificity of neuronotrophic effects. S‐100β and IGF‐II immunoreactivities were expressed in glial cultures derived from the same brain regions, raising the possibility that these factors have autocrine effects on glia as well as paracrine actions on neurons. The results of this study suggest that specificity of neuronotrophic factors for particular embryonic neurons may be correlated with their neurotransmitter phenotype. © Wiley‐Liss, Inc.
Developmental changes in nerve growth factor level in rat serum
Authors: Murase K. , Takeuchi R. , Iwata E. , Furukawa Y. , Furukawa S. , Hayashi K.
Published: January 2005

In serum, nerve growth factor (NGF) forms a complex with α2‐macroglobulin (α2M), which formation inhibits the immunoreactivity between NGF and its antibodies. For measuring the serum level of NGF, it is thus necessary to liberate NGF from the NGF‐α2M complex and prevent reformation of such complex. The pretreatment of rat serum with 1 M guanidine hydrochloride for a few hours and operation of the enzyme immunoassay (EIA) in the presence of guanidine hydrochloride provided a reliable means for determination of the NGF level in serum. By this procedure we followed the serum NGF level in rats developmentally. It increased from prenatal day 2 to postnatal day 5 and decreased slightly at postnatal week 3, thereafter remaining constant throughout adulthood. In pregnant rats, the NGF level in serum increased threefold to fivefold before birth and then decreased rapidly. These data suggest that serum NGF level may reflect the demand for this molecule during establishment of the peripheral nervous system. © Wiley‐Liss, Inc.
Neuromuscular junctions contain NP185: The multifunctional protein is located at the presynaptic site
Authors: Perry D. G. , Li S. , Hanson V. , Puszkin S.
Published: January 2005

The NP185 polypeptide (AP3) is a multifunctional component isolated from brain endocytic vesicles, which binds to tubulin and clathrin light chains, decoated vesicles, synaptic vesicles, and the synaptosomal plasma membrane (Su et al., 1991). The NP185 molecules are expressed during avian cerebellar synaptogenesis and appear to function in CNS regions rich in synaptic terminals (Perry et al., 1991). In this report we describe double‐labelling experiments with avian embryonic striated muscle fibers demonstrating the exclusive presence of the brain‐specific protein at the neuromuscular junction. We used indirect rhodamine immunofluorescence labeling with a monoclonal antibody (mAb‐8G8) to mark the location of NP185 in muscle combined with fluorescein‐alpha‐bungarotoxin to mark the postsynaptic location of the acetylcholine receptors (AChRs). We show that the distribution of both NP185 and AChRs has an overall correlation, but the location of NP185 is circumscribed to presynaptic structures adjacent but not overlapping with postsynaptic structures displaying the AchRs. To confirm the identity of NP185, the molecule was extracted from both tissues, partially purified, immunoprecipitated, and identified in Western blots with the mAb 8G8. The mAb reacted with an identical 185 kD protein band purified from both tissues. Based on its properties and specific neuronal location, the NP185 molecule may function in motor nerve terminals by screening membrane proteins, identifying areas of the synaptic plasma membrane, and to anchor these elements with structural proteins for their recycling and transport within the neuronal cellular compartments. © 1992 Wiley‐Liss, Inc.
Interleukin‐2 inhibits the GABA‐induced Cl‐ current in identified Aplysia neurons
Authors: Sawada M. , Hara N. , Ichinose M.
Published: January 2005

The effects of extracellularly applied recombinant human interleukin‐2 (rhIL‐2) on the γ‐aminobutyric acid (GABA)‐induced Cl‐ current recorded from identified neurons (R9 and R12) of Aplysia kurodai were investigated with conventional voltage‐clamp and pressure ejection techniques. Bath‐applied rhIL‐2 (10–40 U/ml) reduced the GABA‐induced current in the neurons without affecting resting membrane conductance and the holding current. The suppressing effect of rhIL‐2 on the current was completely reversible. Heat‐inactivated rhIL‐2 was without effect. These results suggest that the immunomodulator IL‐2 can modulate the GABA‐induced response in the nervous system. © 1992 Wiley‐Liss, Inc.
Gene therapy of maliganant brain tumors: A rat glioma line bearing the herpes simplex virus type 1‐thymidine kinase gene and wild type retrovirus kills other tumor cells
Authors: Takamiya Y. , Short M. P. , Ezzeddine Z. D. , Moolten F. L. , Breakefield X. O. , Martuza R. L.
Published: January 2005

Tumor cells infected with a retrovirus vector (VIK) containing the herpes simplex virus thymidine kinase (HSV‐TK) gene can be selectively killed by treatment with nucleoside analogues, such as ganciclovir. To mediate delivery of the HSV‐TK gene to “recipient” tumor cells, “donor” C6 rat glioma cells infected with the VIK vector (C6VIK) were superinfected with wild type Moloney murine leukemia virus (WT Mo‐MLV). These modified donor cells (C6VIKWT) produced both wild type retrovirus and the VIK vector. In culture, C6VIKWT cells were 300‐fold more sensitive to the toxicity of ganciclovir than were C6VIK cells, suggesting that the presence of wild type retrovirus contributed to the toxicity. Co‐culture of C6VIKWT cells with the C6 subline, C6BAG, sensitized the latter to ganciclovir treatment. Nude mice inoculated subcutaneously with a mixture of C6VIKWT and C6BAG cells showed regression of subsequent tumors when treated with ganciclovir. The observations show that tumor cells modified in culture by infection with a retrovirus bearing the HSV‐TK gene and wild type retrovirus are not only sensitive to ganciclovir, but can transfer this sensitivity to neighboring “naive” tumor cells in culture and in vivo. © 1992 Wiley‐Liss, Inc.
Expression of the T‐lymphocyte activation gene, F5, by mature neurons
Authors: Arai M. , Prystowsky M. B. , Cohen J. A.
Published: January 2005

F5 was first identified as an mRNA expressed by activated but not resting T‐lymphocytes. Subsequent studies suggested that it also is selectively expressed by mature neurons. Although the F5 protein coding sequence is highly conserved, the function of the F5‐encoded protein is unknown. The present studies were undertaken to define the anatomic distribution, cellular specificity, and developmental pattern of F5 mRNA expression in the mouse nervous system, addressing specifically the question of whether the expression pattern of F5 corresponds to that of known ligand‐receptor or signal‐transduction systems. The use of a nonradioactive in situ hybridization method and paraffin‐embedded sections provided excellent morphological preservation and a high degree of cellular resolution. F5 mRNA was detected in the central nervous system, peripheral nervous system, and retina in cells having the location and morphological features of neurons. Combined in situ hybridization histochemistry for F5 mRNA and immunofluorescence staining for cell‐specific markers confirmed that neurons expressed F5 mRNA but astrocytes did not. The neuronal expression of F5 mRNA had two interesting features. First, the level of expression appeared to correlate directly with the size of the neuronal perikarya, the length of the axonal projection, or the extent of dendritic arborization. Second, F5 mRNA appeared late in post‐natal development. These observations are of interest because of preliminary data suggesting that F5 may function as a substrate for protein kinase C, which demonstrates a similar expression pattern in the nervous system. © 1992 Wiley‐Liss, Inc.