bluuebunny/crossref_metadata_2025 huggingface dataset:
Protection of neuro‐2a cells against calcium ionophore cytotoxicity by gangliosides
Authors: Nakamura K. , Wu G. , Ledeen R. W.
Published: May 2005

Gangliosides are known to assert both neuritogenic and neuroprotective effects when applied to a variety of neuroblastoma and primary neuronal cultures. We have developed a model employing Neuro‐2a Neuroblastoma cells with Ca2+ ionophore A23187 as neurotoxic agent causing neurite retraction and eventual cell death. Gangliosides attenuated the toxicity of this substance, increasing both cell survival and neurite stability. In one series of experiments, cells were exposed to A23187 for 24 hr and then incubated in fresh medium (washout) for 18 hr; gangliosides were present at varying times. The paradigm in which cells were only preincubated (2 hr) with ganglioside provided no benefit, nor did incubation of the cells in both ionophore and ganglioside during the 24‐hr exposure period. Significant protection was achieved by exposing the cells to ganglioside after washout of A23187, or continuously throughout the whole period. Bovine brain ganglioside mixture and the four major components (GM1, GDla, GDlb, GTlb) applied individually were all effective. By contrast, GM3 and GMl‐alcohol, a neutral derivative of GM1, provided little or no protection. Dichlorobenzamil an inhibitor of the Na+ −Ca2+ exchanger, tended to block the neurite stabilizing effect of gangliosides, suggesting that the mechanism might involve potentiation of this antiporter.
Pertussis toxin provides evidence for two independent signalling pathways leading to the activation of the nerve growth factor gene
Authors: Wion D. , Neveu I. , Jehan F. , Brachet P.
Published: May 2005

Increased expression of the nerve growth factor (NGF) gene may be obtained by treating L929 fibroblasts with serum, phorbol 12‐myristate 13‐acetate (PMA), or 1,25‐dihydroxyvitamin D3 (1,25‐(OH)2 D3). The possible involvement of GTP‐binding proteins (G proteins) in these regulatory events was monitored by exposing the cells to pertussis toxin (PT), a compound known to inactivate several types of G proteins by ADP ribosylation. Measurements of the I pool of NGF mRNA by Northern blot analysis, and quantification of the factor secreted by the cells with a double‐site ELISA assay, indicate that pretreatment with PT decreases by about 60% the effect of serum on the levels of NGF transcript and secreted factor. This effect is accompanied by a corresponding decrease of the expression of c‐fos gene, which takes place soon after the addition of serum to the cells. In contrast, PT had no effect on the basal level of NGF mRNA found in cells maintained in serum‐free medium or in cells stimulated with PMA or 1,25‐(OH)2D3. These results indicate that some serum factor(s) acts via plasma membrane receptors able to interact with PT‐sensitive G proteins to modulate NGF gene expression. In contrast, 1,25‐(OH)2D3 appears to mediate its action through a different signalling pathway, which is likely to require its cytosolic receptor, and is independent of PT‐sensitive G protein and c‐fos induction. PMA is thought to directly activate enzymes of the protein kinase C family, and would therefore bypass the step involving activation of these enzymes by a G‐proteindependent pathway. These results suggest that a dys function in the G protein(s) and c‐fos pathway involved in the regulation of NGF gene could lead to neuropathological processes by altering the expression of the NGF gene. In addition, 1,25‐(OH)2 D3, in light of its ability to bypass these pathways, could be of potential interest in the treatment of neurodegenerative diseases.
Sequential effects of astroglial‐derived factors on neurite outgrowth: Initiation by protease inhibitors and potentiation by extracellular matrix components
Authors: Shea T. B. , Beermann M. L. , Nixon R. A.
Published: May 2005

Astroglial‐conditioned medium (GCM) induced two distinct, but intimately related, phases of neuritogenesis in NB2a/dl neuroblastoma cells—a “rapid‐outgrowth,” unstable phase, and a delayed, relatively stable phase, which are apparently regulated by glial‐derived protease inhibitors and laminin, respectively. The initial rapid outgrowth (<4 hr) may be mediated by inhibition of a thrombin‐like protease, present as a serum component and/or adsorbed to the outer neuronal surface, since (1) a similar effect was obtained by serum removal or by adding the specific thrombin inhibitor, hirudin; (2) exogenous thrombin inhibited the rapid outgrowth of neurites by GCM; and (3) cell‐free enzyme assays confirmed the presence of thrombin‐inhibitory activity in GCM. Although neurites induced by removal of serum removal or hirudin addition are rapidly resorbed following serum replenishment or hirudin depletion, GCM‐induced neurites continued to elongate after GCM removal, indicating that GCM contained additional neurite‐promoting factors. Anti‐laminin antiserum did not inhibit the initial elaboration of neurites by GCM but prevented their continued elongation. Anti‐laminin antiserum had no affect on neurite outgrowth induced by serum deprivation. The more protracted, second phase of neurite outgrowth could also be achieved by the addition of soluble purified laminin to undifferentiated cells. Unlike neurites at 4 hr, neurites at 24 hr were no longer dependent on the protease inhibitors in GCM, since exogenous thrombin no longer caused them to retract. Simultaneous addition of thrombin and anti‐laminin antiserum with GCM had identical inhibitory effects on continued neurite elaboration at 24 hr as did anti‐laminin antiserum without thrombin. We conclude that protease inhibitors present in GCM initiate the rapid elaboration of unstable neurites and that glial‐derived extracellular matrix factors support the continued elaboration of these neurites.
Regional distribution of iron and iron‐regulatory proteins in the brain in aging and Alzheimer's disease
Authors: Connor J. R. , Snyder B. S. , Beard J. L. , Fine R. E. , Mufson E. J.
Published: May 2005

It is well established that iron, which is of considerable importance for normal neurological function, is highly regulated in all organ systems. However, until recently, iron regulation in the nervous system has received little attention. This study quantitatively compares the levels of the major iron‐regulatory proteins, transferrin and ferritin, and iron itself in three cerebral cortical regions of the human brain from material collected at autopsy. Three groups were studied: (1) normal adult (under 65 yr of age), (2) aged (> 65), and (3) Alzheimer's disease. Normally, transferrin is more abundant in white matter than in gray matter. Ferritin is approximately 10 x more abundant than transferrin throughout the brain regions examined and is evenly distributed, as is iron, in the gray and white matter. In Alzheimer's disease transferrin is consistently decreased particularly in the white matter of the various cerebral cortical regions examined whereas the iron and ferritin changes are inconsistent. The observations In this study are consistent with our general hypothesis that iron homeostasis is disrupted in the aging brain and the alterations in iron‐regulatory proteins are exacerbated in Alzheimer's disease. The decrease in transferrin levels could indicate a decreased mobility and subsequent utilization of iron in the brain. Such a decrease in iron availability could play a significant role in neuronal degeneration and increased peroxidative damage known to occur in Alzheimer's disease.
Expression of leucocyte adhesion molecules at the human blood‐brain barrier (BBB)
Authors: Rössler K. , Neuchrist C. , Kitz K. , Scheiner O. , Kraft D. , Lassmann H.
Published: May 2005

The expression of leucocyte adhesion molecules was studied on cerebral endothelia by immunocytochemistry. In peritumoral “normal” brain tissue we found low endothelial expression of 1CAMI, LFA3, CD44, and CD9, whereas VLA1 was present on vessels in high incidence and density. LFAl, CD2, and CR3 were found on intraluminal and parenchymal leucocytes, but were absent on brain vessels. In brain tumors and inflammatory brain lesions, we observed an up‐regulation of endothelial ICAM1 and LFA3 expression, whereas other adhesion molecules on endothelial cells remained unchanged.

Within the brain parenchyma, ICAM1 and LFA3 were found on astrocytes and tumor cells; on the contrary, LFA1 was expressed on microglial cells similar to CR3. CD44 and CD9 showed a diffuse neuropil expression in normal and tumoral tissue, whereas VLA1 was not expressed on any parenchymal cells.

Our data show that multiple different adhesion molecules are present on blood‐brain barrier endothelium (BBB) under normal conditions and some adhesion molecules are up‐regulated in brain tumors and under inflammatory conditions. The presence of adhesion molecules in the vessel walls as well as on parenchymal cells like astrocytes and microglia may guide inflammatory cells into and through the brain in the course of immune surveillance and inflammation.
Choline acetyltransferase activity in murine thymus
Authors: Tria M. A. , Vantini G. , Fiori M. G. , Rossi A.
Published: May 2005

Murine thymus has been demonstrated to contain both cholinergic receptors and acetylcholinesterase activity. In the present study we have investigated the presence of the enzyme choline acetyltransferase in this organ, which is responsible for the synthesis of acetylcholine. Results reported here demonstrate that (1) an appreciable amount of the enzyme is already present in the thymus on the day of birth; (2) its expression is developmentally regulated; and (3) thymic atrophy, induced in young (2‐week‐old) and adult (6‐week‐old) mice by i.p. injection of hydrocortisone for 2 days, is accompanied by significant reduction of choline acetyltransferase activity only in young mice.

Altogether these results demonstrate the presence in the murine thymus of functionally relevant markers of the cholinergic system that might interface the interactions between the nervous and immune systems.
Deprenyl reduces the death of motoneurons caused by axotomy
Authors: Salo P. T. , Tatton W. G.
Published: May 2005

Deprenyl, a monoamine oxidase B inhibitor, appears to slow the progression of neurological deficits in Parkinson's disease and cognitive decline in Alzheimer's disease. The mechanisms for the slowing of the diseases are unknown. Deprenyl can reduce the death of murine substantia nigra neurons when administered after the neurons are damaged in MPTP parkinsonism by increasing the neurons' survival after they are damaged, rather than by just protecting the neurons against damage by blocking the conversion of MPTP to its active form as was previously thought. The death, of immature motoneurons after separation from their muscle targets by axotomy provides a model for assessing trophically dependent neuronal survival. To determine whether deprenyl can alter the survival of neurons other than those in the substantia nigra, we examined the survival of rat facial motoneurons after axotomy at 14 days of age. Using a combination of immunocytochemistry for choline acetyl transferase and Nissl staining, we found that deprenyl treatment (10 mg/kg every second day) increased by 2.2 times the number of motoneurons surviving 21 days after the axotomy. This finding showed that deprenyl treatment can rescue neurons other than those in the substantia nigra and can compensate in part for the loss of target‐derived trophic support caused by axotomy.
Effects of acidic fibroblast growth factor on higpocampal long‐term potentiation in fasted rats
Authors: Hisajima H. , Saito H. , Abe K. , Nishiyama N.
Published: May 2005

Effects of human recombinant acidic fibroblast growth factor (haFGF) on long‐term potentiation (LTP) and the increase of the spike amplitude induced by weak tetanic stimulation were investigated and compared with those of CS23 (modified human basic FGF) in the dentate gyrus of fasted and non‐fasted rats. haFGF didn't influence the LTP induced by the tetanus of 100 pulses at 100 Hz in both 24 hr fasted and non‐fasted rats. On the other hand, the tetanus of 20 pulses at 60 Hz significantly enhanced the amplitude of population spike and facilitated the generation of LTP by the i.c.v. injection of 10 μl of 20–40 μg/ml haFGF in 24 hr fasted rats but not in non‐fasted rats. However, 40 μg/ml CS23 induced LTP when the tetanus of 20 pulses at 60 Hz was applied in both fasted and non‐fasted states. These results suggest that haFGF might be one of the regulating factors of feeding and memory.
Acrylamide exposure preferentially impairs axonal transport of glycoproteins in myelinated axons
Authors: Harry G. J. , Morell P. , Bouldin T. W.
Published: May 2005

The right L5 dorsal root ganglion of adult rats exposed to acrylamide (40 mg/kg body weight/day for nine consecutive days) was injected with either [3H]methionine or [3H]glucosamine. After allowing incorporation into macromolecules and axonal transport to proceed for 5 hr, the distribution of radioactivity in cross sections and longitudinal sections of sciatic nerve was determined by autoradiography. Control and treated animals showed no difference in distribution of label within the sciatic nerve with respect to rapidly transported proteins labelled with [3H]methionine. In control animals the distribution of rapidly transported glycoproteins labelled with [3H]glucosamine was similar to that found for [3H]methionine‐labelled proteins. In contrast, acrylamide‐exposed rats had a very different distribution of labelled glycoproteins; there was a marked paucity of label in the myelinated axons. We interpret this result as indicating that acrylamide preferentially inhibits glycosylation or axonal transport of glycoproteins in neurons bearing myelinated axons.
Cyclic AMP‐induced upregulation of proteolipid protein and myelin associated glycoprotein gene expression in C6 cells
Authors: Ye P. , Kanoh M. , Zhu W. , Laszkiewicz I. , Royland J. E. , Wiggins R. C. , Konat G.
Published: May 2005

A Model culture system of C6 rat glioma cells was used to test the involvement of cAMP in the regulation of the myelin PLP and MAG genes. The treatment of cells with isoproterenol (10−5to 10−8M) upregulated the expression of the PLP and MAG genes in a concentration‐dependent manner. The mRNAf or PLP reached a maximum (sevenfold higher than in control cells) after about 12–24 hr, then declined to approximately fourfold over the control level. The response of MAG gene was delayed by at least 36 hr, and the level of MAG mRNA reached a maximum of approximately 48‐fold over the control level on the fourth day in culture. The co‐administration of propranolol blocked the effect of isoproterenol, whereas 10−5M forskolin simulated the effect of isoproterenol, inducating a role of camp in the signal transduction cascades leading to upregulation of the myelin genes. However, the dissimilarity in the timing and the extent of upregulation of the PLP and MAG genes by cAMP‐stimulating agents indicate the existence of different intracellular mechanisms for the activation of these two genes. Cyclocheximide blocked the stimulatory effect of isopoterenol on both the PLP and MAG genes, indicating that the effect of cAMP on the myelin genes is mediated by protein product(s) of other cAMP response gene(s).
Role of amyloid precursor protein (APP): Study with antisense transfection of human neuroblastoma cells
Authors: LeBlanc A. C. , Kovacs D. M. , Chen H. Y. , Villaré F. , Tykocinski M. , Autilio‐Gambetti L. , Gambetti P.
Published: May 2005

The function of amyloid precursor protein (APP) was investigated in human neuroblastoma La‐N‐1 cells by stable transfection with a DNA construct encoding antisense APP mRNA. Levels of APP mRNA, as well as proteins, were reduced by 80–90% in antisense APP transfected (ASAT) cells. ASAT cells exhibited three main features as a result of APP gene expression deprivation: (1) a 30% reduction in cell proliferation, (2) reduced cell adhesion that could be reversed by the addition of La‐N‐1 conditioned media as a source of secreted APP, and (3) a two‐ and four fold increase in neurite‐bearing cells suggesting that; cellular APP may be involved in neurite extension. The first two features confirm previously reported functions for APP in proliferation and adhesion of non‐neuronal cell types but the use of neuroblastoma cells in this study disclose a novel role for cellular APP in neurite extension.
Differentiation of PC12 cells with nerve growth factor is associated with induction of transin synthesis and release
Authors: Fillmore H. L. , Mainardi C. L. , Hasty K. A.
Published: May 2005

We have identified and characterized a calcium‐dependent metalloproteinase which is induced in rat pheochromocytoma cells (PC12 cells) during differentiation with nerve growth factor (NGF). Assays of proteolytic activity in media from differentiated PC 12 cell cultures revealed a NGF‐dependent increase in the activity of a proteinase which has a molecular weight of 62 kDa. Studies using serine, thiol, and metalloproteinase inhibitors demonstrated that the secreted enzyme is a metalloproteinase. Treatment of culture supernatants with aminophenylmercuric acid (APMA), a known activator of metalloproteinases, resulted in a decrease in the molecular weight of the proteinase. Western blot analysis of culture media from NGF‐treated PC12 cells using an antibody directed against a synthetic peptide of rat transin identified this metalloproteinase as transin. Treatment of PC12 cells with acidic and basic fibroblast growth factor (FGF) resulted in distinct morphological changes as well as transin release. Incubation with epidermal growth factor (EGF) did not induce transin release. Dexamethasone inhibited the induction of transin release by NGF. 35S‐methionine labeling and immunoprecipitation of newly synthesized proteins from culture supernatants confirmed that NGF induced the synthesis of this enzyme 8 hr after NGF treatment The NGF‐dependent induction of transin, a calcium‐dependent metalloproteinase which degrades type IV collagen, laminin, and fibronectin suggests that transin may function to degrade the surrounding extracellular matrix during the invasive process of axonal elongation in neuronal development thereby allowing the movement of growth cones and axons toward specific targets.
Novel antigenic determinant expressed in neurons of the dorsolateral hypothalamus in rat and human
Authors: Quinn J. , Eckenstein F. P. , Baughman R. W.
Published: May 2005

Previous studies have identified a group of cells in the dorsolateral hypothalamus that project to many different areas in the CNS, such as thalamus, diagonal band of Broca, basal ganglia, cerebral cortex, hippocampus, and olfactory bulb. Their role is presently unknown, but the cells have been reported to stain for an intriguing array of putative neurotransmitter‐related substances, including α‐melanocyte‐stimulating hormone (αMSH), melanin‐concentrating hormone (MCH), human growth‐hormone‐releasing factor 1–37 (hGRF 1–37), corticotropin‐releasing factor (CRF), metorphamide, and acetylcholine esterase. A monoclonal antibody produced in the present study, αCl1, stains both the cell bodies of this system in hypothalamus, with a punctate pattern, and varicose fibers in the various target areas. In double‐label immunocytochemical experiments in rat DLH, αCl1 and MCH staining exactly overlaps. Concentrations of αMSH and MCH high enough to completely block staining with the corresponding antisera had no effect on staining with αC11. Similarly, CRF, hGRF 1–37, and metorphamide were unable to block αCl1 staining. The results suggest that the antigenic epitope for αCl1 is not contained in αMSH, MCH, CRF, hGRF, or metorphamide, and thus, that αCl1 is detecting another antigen uniquely expressed in these neurons. The punctate appearance of staining in the hypothalamus and the concentration of staining in fiber varicosities suggests that the αCl1 epitope may be involved in synaptic function.
Bimodal regulation of protein phosphorylation by a ganglioside in mat brain membrane
Authors: Nakaoka T. , Tsuji S. , Nagai Y.
Published: May 2005

Whether or not a ganglioside influences the protein phosphorylation in the rat brain membrane fraction was investigated. Phosphorylation of the 72 kDa protein was significantly affected by the addition of 80 nM GQlb in vitro, which is far below the reported concentration of gangliosides that affects protein phosphorylation in the neuronal membrane fraction. This action of GQlb was bimodal: it being not only stimulatory as to the incorporation of phosphate into the 72 kDa protein on incubation for 20 sec, but also as to the release of phosphate from or breakdown of the 72 kDa protein on incubation for more than 5 min. Eighty nM GQlb did not noticeably affect ATPase in the same fraction. These results suggest that the transphosphorylation of the 72 kDa protein is affected by the interaction of GQlb with either the responsible enzymes or the 72 kDa protein as a substrate.
Establishment and characterization of a mouse Schwann cell line which produces myelin in vivo
Authors: Boutry J.‐M. , Hauw J.‐J. , Gansmüller A. , Di‐Bert N. , Pouchelet M. , Baron‐Van Evercooren A.
Published: May 2005

A Schwann cell line (MSC 80) was established from purified mouse Schwann cell cultures using large doses of serum. MSC 80 cell line is an aneuploid cell line which has a doubling time of 17 hr and has been maintained through more than 110 passages. Most of MSC 80 cells are of bipolar or stellate (3–5 processes) shape. A few others are irregular in shape, gigantic, and multinucleated. All MSC 80 cells express antigens of myelin‐forming Schwann cells such as S‐100, 224/58, laminin, and other glycoproteins of the extracellular matrix. However, they also express the nonmyelin‐forming Schwann cell antigen GFAP. By time‐lapse cinematography, MSC 80 cells exhibit the Schwann cell characteristic rhythmical undulations. When induced to form aggregates in agar, they form intercellular junctions and basement membrane‐like structures. In addition, after transplantation in or at a distance from a lysolecithin induced lesion, MSC 80 cells from myelin around the host demyelinated axons. MSC 80 cells thus express, when isolated in vitro, some of the normal myelin‐forming Schwann cell phenotype. In addition, they present the major advantage of forming myelin when associated with axons in vivo. © 1992 Wiley‐Liss, Inc.
Cloning of cDNAs encoding human S‐100 α and β subunits and their differential expression in human tumor cell lines
Authors: Morii K. , Tanaka R. , Takahashi Y. , Kuwano R.
Published: May 2005

We isolated nearly full‐length clones of S‐100 α and β subunit cDNAs from a human brain cDNA library. The α subunit cDNA was 579 bp long and contained 99 bp of 5′‐noncoding region, 282 bp of coding region, and 198 bp of 3′‐noncoding region. The β subunit cDNA was 743 bp long and contained 57 bp of 5′‐noncoding region, 276 bp of coding region, and 410 bp of 3′‐noncoding region. An amino acid sequence comparison between human and bovine α subunits and between human and rat β subunits showed that both subunits were nearly entirely conserved. The amino acid sequences of human α and β subunits were conserved at both Ca2+‐binding domains.

Northern blot analysis of brain RNA showed that human α and β subunit cDNA probes discriminated between α and β subunit mRNAs. By using these subunit‐specific cDNA probes, it was demonstrated that α and β subunit mRNAs were expressed in different manners among tumor cell lines: β was detected in melanoma and some glioma cell lines, while α was detected only in a melanoma cell line. Southern blot analysis showed that there were no major deletions and rearrangements of α and β subunit genes in these cell lines, regardless of the level of α and β subunit expression, suggesting that the expression of these subunits may be regulated at the transcriptional or RNA stability level. © 1992 Wiley‐Liss, Inc.
Synaptosomal plasma membrane transport of excitatory sulphur amino acid transmitter candidates: Kinetic characterisation and analysis of carrier specificity
Authors: Grieve A. , Butcher S. P. , Griffiths Roger
Published: May 2005

The transport kinetics of the excitatory sulphur‐containing amino acid (SAA) transmitter candidates, L‐cysteine sulphinate (L‐CSA), L‐cysteate (L‐CA), L‐homocysteine sulphinate (L‐HCSA), and L‐homocysteate (L‐HCA), together with their plasma membrane carrier specificity, was studied in cerebrocortical synaptosome fractions by a sensitive high performance liquid chromatographic assay. A high affinity uptake system could be demonstrated for L‐CSA (Km =; 57 ± 6 μM; Vmax =; 1.2 ± 0.1 nmol/min/mg protein) and L‐CA (Km =; 23 ± 3 μM; Vmax =; 3.6 ± 0.1 nmol/min/mg protein), whereas L‐HCSA (Km =; 502 ± 152 μM; Vmax =; 6.1 ± 1.3 nmol/min/mg protein) and L‐HCA (Km =; 1550 ± 169 μM; Vmax =; 10.3 ± 1.1 nmol/min/mg protein) exhibited much lower affinity as transport substrates. In all cases, only a single, saturable Na+‐dependent component of uptake could be identified, co‐existing with a non‐saturable, Na+‐independent influx component. Plasma membrane carrier specificity of the SAAs was established following comparison with other high‐affinity neurotransmitter systems. High‐affinity L‐CSA and L‐CA transport and low‐affinity L‐HCSA and L‐HCA transport demonstrate strong positive correlations in inhibition profiles when compared against each other or individually against the high‐affinity transport of L‐[3H]glutamate, L‐[3H]aspartate, or D‐[3H]aspartate. Moreover, the transport systems for the excitatory SAAs exhibited a negative correlation when compared in inhibition profiles with the high affinity transport of both [3H] γ‐aminobutyric acid (GABA) and [3H]taurine. Taken together, these results strongly suggest that L‐CSA, L‐CA, L‐HCSA, and L‐HCA share a common synaptosomal plasma membrane transport system with L‐glutamate, L‐aspartate, and D‐aspartate. © 1992 Wiley‐Liss, Inc.
Neuronal protein tyrosine kinases associated with synaptosomal glycoproteins
Authors: Hanissian S. H. , Sahyoun N.
Published: May 2005

Protein tyrosine kinase (PTK) activity associated with synaptosomal membrane glycoprotein (SMGP) fractions of rat brain was examined. The synthetic substrate poly(Glu4‐Tyr) was phosphorylated by SMGP in the presence of Mg2+ and Mn2+, whose stimulatory effects were additive. In contrast, endogenous tyrosine phosphorylation in SMGPs was strictly dependent on Mn2+. Anti‐phosphotyrosine antibodies (PY20) immunoprecipitated two polypeptides in SMGPs of Mr 170K and 60K. Upon preincubation with IGF‐I, 97/90K polypeptides were phosphorylated, corresponding to the IGF‐I receptor beta‐subunits, and were immunoprecipitated with both PY20 and anti‐IGF‐I‐receptor antibodies. Immunoblot analysis using anti‐src antibody revealed that there was src protein associated with the glycoprotein fractions of solubilized synaptosomal membranes. Additional experiments revealed that the 60K tyrosine‐phosphorylated polypeptide present in the PY20 precipitates was indeed pp60 c‐src. This was confirmed by subjecting the PY20 immunoprecipitates to immunoblotting using anti‐src antibodies. In addition, src protein was directly immunoprecipitated by anti‐src antibodies from the SMGP preparations. Hence, IGF‐I receptors and glycoprotein‐associated PTKs in cluding pp60c‐src may play an important role in synaptic transmembrane signalling, plasticity, and neuronal survival. © 1992 Wiley‐Liss, Inc.
Phorbol esters modulate phospholipid metabolism in a human cholinergic cell line, LA‐N‐2: A possible role for the base exchange enzymes
Authors: Singh I. N. , McCartney D. G. , Sorrentino G. , Massarelli R. , Kanfer Julian N.
Published: May 2005

It was shown that in LA‐N‐2 cells prelabeled with [3H‐methyl]choline for 24 hr (Singh et al.: Mol Chem Neuropathol 14:53–66, 1991) the major intracellular and extracellular hydrophilic compound was phosphorylcholine. LA‐N‐2 cells were labeled with [14C‐methyl]choline for 24 hr, harvested, and incubated in Hepes/BSA/saline buffer for varying periods of time. The radioactive compound present in the cytosol and released into Hepes/BSA/saline buffer medium in the presence or absence of TPA was phosphorylcholine. There was a gradual increase in the appearance of radioactivity in the medium and this corresponded to a gradual decline in the radioactivity present in the cytosolic compartment with a statistically significant P value of <.005. Identical results were obtained with prelabeled cells subsequently incubated with TPA. There was no significant change in the amount of radioactivity associated with lipid suggesting that the phosphorylcholine may be released directly from the cytosolic compartment into the medium rather than originating through a phospholipase‐C catalyzed hydrolysis of phosphatidylcholine. This possibility received support from experiments in which cells were electropermeabilized in the presence of radioactive phosphorylacholine. It was found that the introduced [14C]phosphorylcholine was released intact into the incubation medium from the cytosolic compartment.

The incorporation of [14C]choline, [14C]ethanolamine, and [14C]serine by LA‐N‐2 cells into their corresponding phospholipids was investigated in the presence or absence of TPA. The presence of TPA increased the amount of radioactivity incorporated into the phospholipids with a corresponding decrease in the amount of radioactivity in the cytosolic compartment compared to control cultures. There were no detectable differences between TPA exposed and control cells in the distribution of radioactivity in free choline, phosphorylcholine, or CDP‐choline of [14C] choline labeled cells. This indicates that the increased lipid labeling was not accompanied by enhanced labeling of the intermediates of the de novo pathway. This effect of TPA in altering the distribution of labeling of the cytosolic and lipid components was not demonstrable with cells grown in the presence of 10−5M retinoic acid. © 1992 Wiley‐Liss, Inc.
Ionic channel currents in cultured neurons from human cortex
Authors: Simard J. M. , Song Y. , Tewari K. , Dunn S. , Werrbach‐Perez K. , Perez‐Polo J. R. , Eisenberg H. M.
Published: May 2005

Ionic channels in human cortical neurons have not been studied extensively. HCN‐1 and HCN‐1A cells, which recently were established as continuous cultures from human cortical tissue, have been shown by histochemical and immunochemical methods to exhibit a neuronal phenotype, but expression of functional ionic channels was not demonstrated. For the present study, HCN‐1 and HCN‐1A cells were cultured in Dulbecco's modified Eagle's medium with 15% fetal calf serum, in some cases supplemented with 10 ng/ml nerve growth factor, 10 μM forskolin, and 1 mM dibutyryl cyclic adenosine monophosphate to promote differentiation. Cells or membrane patches were voltage clamped using conventional patch clamp techniques. In HCN‐1A cells, we identified a tetrodotoxin‐sensitive Na+ current, two types of Ca2+ channel current, including L‐type current and a second type that in some respects resembled N‐type current, and four types of K+ current, including a delayed outward rectifier that showed voltage‐dependent inactivation, two types of noninactivating Ca2+‐activated K+ channels with slope conductances of 146 and 23 pS (K+ iK+ o 145 mM/5 mM), and less frequently, a noninactivating, intermediate conductance channel that was not sensitive to internal Ca2+. When HCN‐1A cells were examined after 3 days of exposure to differentiating agents, pronounced morphological changes were evident but no differences in ionic currents were apparent. HCN‐1 cells also exhibited K+ and Ca2+ channel currents, but Na+ currents were not detected in these cells. Our data provide additional evidence indicating a functional neuronal phenotype for HCN‐1A cells, and represent the most comprehensive survey to date of the variety of ionic channels expressed by human cortical neurons. © 1993 Wiley‐Liss, Inc.
Identification of neuropeptide Y receptors in cultured astrocytes from neonatal rat brain
Authors: Gimpl G. , Kirchhoff F. , Lang R. E. , Kettenmann Helmut
Published: May 2005

Specific binding sites for neuropeptide Y could be demonstrated in primary cultures of astrocytes from neonatal rat brain. Neuropeptide Y binding was saturable, reversible, and temperature dependent as revealed by saturation studies and kinetic experiments. Scatchard analysis of equilibrium binding data indicated a single population of high‐affinity binding sites with respective KD and Bmax values of 0.43 nM and 6.9 fmol/2.7 × 105 cells. Physiological responses induced by neuropeptide Y could be detected in a distinct subpopulation of cultured astrocytes on the basis of two criteria: (1) electrophysiological responses and (2) single cell measurements of changes in [Ca2+]i. In that fraction of cells responding (20–70%, varying among cultures from different preparations), brief application of neuropeptide Y led to a membrane potential depolarization, lasting several minutes. When the membrane was clamped close to the resting membrane potential using the whole‐cell patch‐clamp technique, neuropeptide Y induced an inward current with a similar time course as the neuropeptide Y‐induced membrane depolarization. As detected by single cell microfluorimetric (fura‐2) measurements neuropeptide Y induced an increase of [Ca2+]i which was caused by the entry of extracellular Ca2+. Both the [Ca2+]i increase and the electrophysiological responses were unaffected by pretreatment of the astrocytes with pertussis toxin. © 1993 Wiley‐Liss, Inc.
Molecular biology of vertebrate learning: Is c‐fos a new beginning?
Authors: Kaczmarek L.
Published: May 2005

An elevated expression of c‐fos nuclear protooncogene (immediate early gene) has been shown repeatedly to correlate with long‐term neuronal responses. This paper reviews recent data suggesting that neuronal plasticity, including learning and memory formation, may involve c‐fos expression as well. On the basis of biological functions of the Fos protein, well known to be a component of a transcription factor, AP‐1, (activator protein 1) a hypothesis suggesting a role of transcription factors in the integration of information during learning processes is proposed. © 1993 Wiley‐Liss, Inc.
FGF‐2‐mediated protection of cultured mesencephalic dopaminergic neurons against MPTP and MPP+: Specificity and impact of culture conditions, non‐dopaminergic neurons, and astroglial cells
Authors: Otto Dörte , Unsicker K.
Published: May 2005

The protective role of basic fibroblast growth factor (FGF‐2) for 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP)‐ and methylpyridiniumion (MPP+)‐lesioned dopaminergic (DAergic) nigrostriatal neurons was studied, using dissociated cell cultures of embryonic day (E) 14 rat mesencephalon. Cells were grown in different culture media and received FGF‐2 (5 ng/ml) and/or the toxins (5 μM) at various schedules, but were consistently allowed to differentiate for 3 days prior to becoming exposed to the toxin. Survival of tyrosine hydroxylase (TH)‐immunoreactive cells at 7 days was only markedly impaired by MPTP, if horse serum (HS) or bovine serum albumine (BSA) were omitted from the culture medium. FGF‐2 increased the number of TH‐immunoreactive cells, and this increase not diminished by MPTP under any culture condition. Uptake of 3H‐DA was significantly reduced by MPTP in HS‐ and BSA‐containing, but not in protein‐less cultures. A protective effect by FGF‐2 was only seen in the presence of BSA. MPP+ caused a more pronounced reduction in 3H‐DA uptake than MPTP, and this effect was partially reversed by the addition of FGF‐2, unless cultures contained HS. Neurofilament protein (NF), an indirect measure for the total number of neurons present in the cultures, was not significantly reduced by MPTP or MPP+ corroborating the specificity of the toxin for DAergic neurons, which constitute only a minor fraction in these cultures. In line with the wide spectrum of target neurons of FGF‐2, this factor significantly increased NF contents under any culture condition. Quantification of the amounts of glial fibrillary acidic protein (GFAP) revealed stimulatory effects of FGF‐2 (2.5‐ to 4‐fold) and at least 10‐fold higher levels in the presence as compared to the absence of HS. These data show that FGF‐2 can protect DAergic neurons against MPTP‐ and MPP+‐mediated damage. However, the effects of the toxins as well as of FGF‐2 are partially dependent on culture conditions. Variations in the effectiveness of toxins and FGF‐2 are not overtly related to the total numbers of neurons or astroglial cells, but may reflect culture type‐dependent alterations of neuronal and glial metabolism. © 1993 Wiley‐Liss, Inc.
Reactive astrocytes involved in the formation of lesional scars differ in the mediobasal hypothalamus and in other forebrain regions
Authors: Alonso G. , Privat A.
Published: May 2005

The fine organization of lesional scars was studied in adult rats at the level of 2 types of surgical cuts aimed at deafferentating the dorsal hypothalamus from its neuropeptide‐Y innervation. These included (i) lesions located dorsolateral to the dorsal hypothalamus, which were shown to form a permanent obstacle to the regeneration of transected neuropeptide‐Y‐fibers, and (ii) lesions located in the ventromedial hypothalamus, where transected neuropeptide‐Y‐fibers were shown to penetrate and eventually cross the lesional area. Double labeling immunocytochemistry and conventional electron microscopy were used to identify various molecules produced by reactive astrocytes and to visualize their ultrastructural organization within the scars, respectively. In the different portions of the dorsolateral scars, the large majority of reactive astrocytes was characterized by a strong immunoreactivity to glial fibrillary acidic protein, vimentin, and embryonic (polysialilated) NCAM. Intense laminin‐immunoreactivity was also observed over large patches included in the scar. Electron microscope observations further indicated that the matrix of the scar was mainly composed of tightly packed astrocytic perikarya and processes connected by extended gap junctions. All around the extracellular and perivascular spaces, these astrocyte profiles were bordered by a thick basal lamina. Only scarce axonal profiles were detected in the core of the scar, most of which exhibited degenerative features. In the ventromedial hypothalamic scars, reactive astrocytes were found to exhibit intense immunoreactivity to both glial fibrillary acidic protein and vimentin. On the other hand, only slight immunostaining to embryonic NCAM and laminin were associated with this type of lesional scar. At the ultrastructural level, the main differences with the dorsolateral scars concerned (i) the gap junctions, which were less frequent and involved shorter portions of adjacent membranes; (ii) the basal lamina, which was essentially localized to the perivascular spaces; and (iii) the axonal profiles, which were frequently observed throughout the scar matrix. These data indicate that reactive astrocytes that formed the glial scar differ in the mediobasal hypothalamus and in other forebrain regions. This provides strong support for the hypothesis that the regeneration of neuropeptide‐Y axons through a mediobasal hypothalamic surgical cut depends mainly on the particular organization of the astroglial scar. © 1993 Wiley‐Liss, Inc.
Rat cerebral cortical neurons in primary culture release a mitogen specific for early (GD3+/04−) oligodendroglial progenitors
Authors: Hardy R. , Reynolds Richard
Published: May 2005

The development of cells of the oligodendroglial lineage, from immature progenitor to myelinating cells, occurs largely in a neuronal environment, yet little is known about specific interactions between these 2 cell types. We have tested the effects of medium conditioned by cultures of rat cerebral cortical neurons (CCM), cerebellar granule interneurons (GCM), and a dorsal root ganglion derived cell line (NDCM) on cells of the oligodendroglial lineage in culture. Different stages of the lineage were defined using the cell surface antigens GD3, 04, and GalC. CCM and NDCM were mitogenic for the early GD3+/04− oligodendroglial progenitor, whereas GCM was mitogenic for the later GD3+/04+ stage. Neutralising antibodies to PDGF and bFGF were able to eliminate the mitogenic activity of NDCM and GCM, respectively, but did not abolish the mitogenic effect of CCM. We have demonstrated that neurons in primary culture from distinct CNS regions exert diffent influences on cells of the oligodendroglial lineage, and specifically that cortical neurons release an unknown mitogen for GD3+/04− oligodendroglial progenitors. © 1993 Wiley‐Liss, Inc.
Alteration of carnosine expression in olfactory system of mouse after unilateral naris closure and partial bulbectomy
Authors: Sakai M. , Nagatsu I.
Published: May 2005

To investigate the function of the dipeptide carnosine in the olfactory system, alterations of carnosine in the primary olfactory nerve after unilateral naris closure or olfactory bulb semilesion were studied by means of immunocytochemistry. After unilateral naris closure, carnosine staining showed no alteration, but immunoreactivity of tyrosine hydroxylase (TH), the rate‐limiting enzyme for biosynthesis of dopamine in dopaminergic neurons, decreased dramatically in periglomerular neurons. This behavior of carnosine is consistent with that reported for the olfactory marker protein (OMP). On the other hand, following partial lesion of the olfactory bulb, ordinary to strong carnosine expression was seen in newly innervated glomeruli that exceeded that seen in the conventional targets. Ectopic, TH‐positive, periglomerular cell‐like neurons appeared around the newly formed glomeruli. These data suggest that carnosine may have some role in regulating TH expression in post‐synaptic neurons. © 1993 Wiley‐Liss, Inc.
Association of glucocorticoid receptor immunoreactivity with cell membrane and transport vesicles in hippocampal and hypothalamic neurons of the rat
Authors: Liposits Zs. , Bohn M. C.
Published: May 2005

The aim of the present study was to reveal at the ultrastructural level cytoplasmic loci that display glucocorticoid receptor (GR) immunoreactivity in pyramidal neurons of the CA1 sector of the hippocampus and in cells of the medial parvicellular subnucleus of the hypothalamic paraventricular nucleus (PVN). Adrenalectomized male rats were injected intraperitoneally with corticosterone (CS) (1 mg/100 g bw) and sacrificed within 4 hr. Vibratome sections of the perfusion‐fixed forebrains were processed for immunocytochemical detection of type 2 GR by means of the BuGr, anti‐rat liver GR monoclonal antibody and silver‐gold‐intensified diaminobenzidine chromogen. The corticosterone administration gradually shifted the GR immunoreactivity (IR) from the cytoplasm to the nucleus. Samples taken 20–40 min after the steroid treatment demonstrated pyramidal cells expressing GR IR in both the cytoplasmic and nuclear pools. Although the chromatin‐associated appearance of GR in the nucleus was identifiable at the light microscopic level, the nature of immunoreactive intracytoplasmic loci was not. Ultrastructural analysis of the cytoplasm indicated that fine silver‐gold grains marking GR‐immunoreactive sites associated with the plasma membrane and coated and regular vesicles. Noted occasionally beneath the plasma membrane of the cell bodies and dendrites, the vesicles also appeared at deeper locations in dendritic processes and around the cell nuclei.

These results suggest that glucocorticoid receptors participate in signal transduction at the level of the cell membrane, as well as at the level of the genome in the cell nucleus. Published 1993 by Wiley‐Liss, Inc.
Effects of nerve growth factor on the survival of primary cultured adult and aged mouse sensory neurons
Authors: Jiang Z.‐G. , Smith R. A.
Published: May 2005

This study investigated the effects of exogenous nerve growth factor (NGF) on the survival and differentiation in primary culture of sensory neurons isolated from adult (6 months) and aged (2 years) mice. For neurons prepared from adult mice, a concentration effect was evident during a 2 week culture period: Neuronal counts in cultures supplemented with 25 and 50 ng/ml NGF did not differ significantly from those of control cultures without exogenous NGF or those with anti‐NGF included in the culture medium, whereas cultures supplemented with either 100 or 200 ng/ml NGF contained higher numbers of neurons throughout the culture period. Cultures prepared from aged mice contained less neurons than those from adult mice, although those supplemented with 100 ng/ml NGF retained higher neuronal numbers than cultures from aged mice which did not receive exogenous NGF. Neuronal diameters were measured to investigate whether specific subpopulations of neurons were more dependent on NGF; the results indicate that neurons of a medium‐larger diameter were more prevalent than cells with a smaller diameter following NGF administration. A shape index was calculated for each culture regimen; with longer culture periods a higher proportion of spindle‐shaped neurons was observed. © 1993 Wiley‐Liss, Inc.
Identification of membrane‐bound carbonic anhydrase in white matter coated vesicles: The fate of carbonic anhydrase and other white matter coated vesicle proteins in triethyl tin‐induced leukoencephalopathy
Authors: Sapirstein V. S. , Durrie R. , Nolan C. E. , Marks N.
Published: May 2005

We have extended our studies on the content of white matter derived coated vesicles (WMCVs) to show that they are enriched in membrane‐bound carbonic anhydrase. Within the myelin complex membrane‐bound carbonic anhydrase is concentrated in the periaxolemmal domain; however, this protein is enriched almost sevenfold in the bilayer of coated vesicles even relative to this myelin membrane region. These data suggest that some vesicles are derived from a site at which this enzyme is highly localized. The enrichment observed for membrane‐bound carbonic anhydrase is unique since other periaxolemmal proteins such as CNPase and plasmolipin are only present in equal amounts in periaxolemmal‐myelin fractions and WMCVs. Based on their known localization, the presence of CNPase coupled with the absence of MAG in WMCVs suggest that these vesicles are derived from the paranodal region. The identification in WMCVs of periaxolemmal‐myelin proteins associated with ion and fluid movement, such as carbonic anhydrase, Na+, K+ ATPase, and the putative K+ channel protein plasmolipin, prompted us to examine the status of these vesicles in triethyl tin (TET)‐induced myelin edema. Coated vesicles and other membrane fractions were isolated from whole brains of control and TET‐treated rats. Whole brains were used so we could compare the effects of TET on WMCV proteins with the effect on proteins enriched in gray matter coated vesicles. The results indicated that TET had no detectable effect on compact or periaxolemmal‐myelin, however, Western blot analysis showed that WMCV proteins, such as carbonic anhydrase, CNPase, and plasmolipin, were virtually absent or greatly diminished from the whole brain coated vesicle fraction. In contrast, gray matter coated vesicle proteins such as synaptophysin, SV2, and the general vesicle protein, cation‐independent mannose‐6‐phosphate receptor were unchanged. These results indicate that coated vesicles may be a useful probe for membrane changes associated with leukopathologies. © 1993 Wiley‐Liss, Inc.