bluuebunny/crossref_metadata_2025_split huggingface dataset:
Po mRNA expression in cultures of Schwann cells and neurons that lack basal lamina and myelin
Authors: Brunden K. R. , Brown D. T. 
Published: January 2005

AbstractSchwann cells of the peripheral nervous system depend on the presence of both axons and basal lamina to achieve a myelinating phenotype. Furthermore, removal of axonal influence results in the cessation of myelination and down‐regulation of myelin protein expression by Schwann cells. Here we examine whether both axons and basal lamina are required by Schwann cells for the expression of mRNA encoding the major myelin glycoprotein, Po. Cultures of Schwann cells and neurons obtained from dorsal root ganglia of 15 day embryonic rat pups were grown for up to 20 days in vitro under conditions that either allowed or prohibited basal lamina and myelin formation. These cultures were assayed for the expression of Po. mRNA by using an S1 nuclease‐protection assay. After 20 days in vitro, the cultures that did not assemble basal lamina and that were incapable of myelin formation expressed Po. mRNA at a level which was comparable to that seen in identically aged, myelinating cultures. Both the myelinating and nonmyelinating cultures demonstrated an appreciable increase in Po mRNA when compared to the starting embryonic dorsal root ganglia Schwann cells. The latter had a low, but detectable, level of mRNA for this myelin glycoprotein. The cultures that were devoid of basal lamina and myelin showed a clear increase in Po mRNA by 11–15 days in culture. This increase in expression depended on the presence of neurons/neurites, since Schwann cells which were grown in neuron‐depleted cultures expressed little, if any, Po mRNA. In contrast to the levels of Po. mRNA, the nonmyelinating cultures had a significantly lower amount of Po. glycoprotein than did the cultures which assemble myelin. This suggests that the nonmyelinating Schwann cells regulate the level of this glycoprotein at the translational and/or the posttranslational level. The data presented here suggest that myelin protein mRNA expression and myelin assembly by Schwann cells are separable events, with the former depending on one or more neuronal/axonal factors.
Phosphorylation of the glial fibrillary acidic protein
Authors: Noetzel M. J. 
Published: January 2005

AbstractThe glial fibrillary acidic protein (GFAP) was found to be phosphorylated in vivo after intracerebral injection of [32P]‐orthophosphate, in brain Slices, and in a cell free system. The phosphorylated proteins were separated by two‐dimensional gel electrophoresis and then transferred to nitrocellulose sheets. Two isoelectric variants of GFAP were immunochemically identified by monoclonal antibodies. Autoradiography demonstrated that only the more acidic isoelectric variant of GFAP was phosphorylated. Phosphoamino acid analysis revealed that under all conditions GFAP was phosphorylated at serine and threonine residues. Incubation of brain slices with[32P]‐orthophosphate and the protein kinase C activator phorbol 12‐myristate 13‐acetate or forskolin, an activator of cyclic AMP‐dependent protein kinase, stimulated phosphorylation of GFAP. Likewise phosphorylation of GFAP was also accentuated by calcium/phosphatidylserine/diolein and by exogenous cyclic AMP‐dependent kinase in a cell free system. These findings announce that protein kinase C and cyclic‐AMP dependent kinase may play physiologic roles in the in situ phosphorylation of GFAP. When isolated cytoskeletal preparations were incubated with [γ‐32P] ATP, GFAP was phosphorylated in vitro by two additional protein kinases, a Ca++/calmodulin‐depen‐dent kinase and an effector‐independent kinase. The results of these investigations strongly suggest that phosphorylation of GFAP appears to be regulated by multiple second messenger pathways.
Recombinant peripheral myelin protein Po confers both adhesion and neurite outgrowth‐promoting properties
Authors: Schneider‐Schaulies J. , von Brunn A. , Schachner M. 
Published: January 2005

AbstractTo probe into the functional properties of the major peripheral myelin cell surface glycoprotein Po, its ability to confer adhesion and neurite outgrowth‐promoting properties was studied in cell culture. To this aim, Po was expressed as integral membrane glyco protein at the surface of CV‐1 cells with the help of a recombinant vaccinia virus expression system. Furthermore, the immunoglobulin‐like extracellular domain of Po (Po‐ED) was expressed as soluble protein in a bacterial expression system and used as substrate coated to plastic dishes or as competitor in cell adhesion and neurite outgrowth‐promoting assays. The adhesion of Po‐expressing CV‐1 cells to Po‐ED substrate was specifically inhibitable by polyclonal Po antibodies (54% ± 6%). In addition, the specific interaction between Po molecules could be reduced (49% ± 8%) by adding soluble Po‐ED to the culture medium, demonstrating that the homophilic interaction between recombinant Po molecules can be mediated, at least on one partner of interacting molecules, by the unglycosylated Ig‐like domain. Substrate‐coated Po‐ED also conferred adhesion and neurite outgrowth ability to dorsal root ganglion neurons with neurites of a mean length of about 150 μm. This neurite outgrowth was specifically inhibitable by soluble Po (74% ± 14%) and Po antibodies (65% ± 9%). These observations indicate that Po is capable of displaying two different types of functional roles in the myelination process of peripheral nerves: The heterophilic interaction with neurons may be responsible for the recognition between axon and myelinating Schwann cell at the onset of myelination, whereas the homophilic interaction may indicate its role in the self‐recognition of the apposing loops of Schwann cell surface membranes during the myelination process and in the mature compact myelin sheath.
Phagocytosis of peripheral nerve myelin in vitro: Effect of antibody
Authors: Smith M. E. , Sadler R. H. , Dyer C. , Benjamins J. A. , Yu A. C. H. 
Published: January 2005

AbstractWe have previously shown that antisera to whole CNS myelin, whole PNS myelin, galactocerebroside (GC), and myelin basic protein (MBP) promote the uptake of CNS myelin by cultured macrophages, and stimulate the conversion of myelin lipids to cholesterol ester and triglycerides. Here we report the results of similar studies using PNS myelin purified from the rat sciatic nerve. Antisera to whole CNS myelin, whole PNS myelin, GC, and MBP preincubated with 14C‐labeled PNS increased the production of radioactive cholesterol ester by macrophages in culture to a level about twice that with preimmune serum, and five to six times that of untreated myelin. The amounts of [14C]triglyceride were similarly increased with these antisera, whole Po and P2 antisera had little or no effect. IgG prepared from the antisera stimulated lipid metabolism to al most the same extent, while heating the antisera did not decrease the stimulatory effect, indicating that myelin was opsonized by IgG, but not likely by complement. With a few exceptions, the four active sera and their IgGs promoted the macrophage metabolism of CNS and PNS myelin almost equally. The cultured macrophages converted about 3% of untreated CNS myelin and about 6% PNS myelin cholesterol to cholesterol ester. Under phase contrast microscopy it was noted that vesicles of CNS myelin appeared to bind individually to macrophages, whereas PNS myelin vesicles tended to self‐associate to form large clumps which were bound to macrophages. Binding studies showed PNS myelin to be bound more firmly to macrophages than CNS myelin. The presence of high amounts of glycoprotein in PNS myelin may account for this difference in binding to macrophages, either through self‐adhesion of the PNS myelin particles, or by the presence of carbohydrate receptors in macrophages. These results may define a role for antibody in myelin destruction in cell‐mediated demyelinative diseases, and may be relevant to the differences seen in vivo between the rates of demyelination of peripheral and central nerves.
Differential regulation of peripherin and neurofilament gene expression in regenerating rat DRG neurons
Authors: Wong J. , Oblinger M. M. 
Published: January 2005

AbstractQuantitative in situ hybridization and RNA blotting methods were used to define the time course and magnitude of changes in expression of mRNAs encoding peripherin and the neurofilament (NF) triplet proteins in rat dorsal root ganglion (DRG) neurons during axonal regeneration. mRNA levels in adult rat L4 and L5 DRGs were examined in autoradiograms after in situ hybridization with specific 35S‐labeled cDNA probes 1–56 days following unilateral crush lesions of the sciatic nerve. The results of quantitative analyses indicated that peripherin mRNA levels were significantly increased in large‐sized (>1000 μm2) DRG neurons at 7, 14, and 28 days after axotomy while the mRNA levels for each of the NF triplet proteins were significantly decreased at these same time points. The mRNA levels of the low (NF‐L) and middle (NF‐M) sized NF subunits were significantly decreased as early as 1 day postaxotomy but the mRNA level of the large NF subunit (NF‐H) did not change until 7 days after axotomy. The maximal reduction in NF mRNA levels was observed at 14 days postaxotomy when NF‐L mRNA levels were only 35% of those in large‐sized, normal control neurons. Recovery toward normal levels of both NF and peripherin mRNAs was observed at 8 weeks postaxotomy. RNA blot analyses with total RNA obtained from DRGs at different postaxotomy times confirmed that NF‐L mRNA levels were reduced in the DRG during the first 4 weeks after axotomy but, interestingly, failed to detect an increase in peripherin mRNA levels. This difference concerning peripherin mRNA levels in axotomized preparations obtained by RNA blotting vs. in situ hybridization was attributed to the fact that RNA blots utilized total DRG RNA which includes mRNAs from both small and large‐sized DRG neurons. A recent in situ hybridization study showed that the small‐sized DRG neurons which contain the majority of the peripherin mRNA in the DRG do not increase their peripherin mRNA levels 14 days after axotomy (Oblinger et al., 1989b). This may mask any change in the large neuron response when total RNA is examined. Overall, the results of this study demonstrate (1) that type III (peripherin) and type IV (NF) intermediate filament genes are regulated differently during axonal regeneration, and (2) that the three NF genes are down‐regulated in a fairly coordinate manner during regeneration. These data suggest that an important component of the regeneration program is the alteration of the composition of the IF component of the cytoskeleton. A cytoskeleton with fewer Type IV IFs and more Type III IFs may be more compatible with the specific structural and functional requirements of regenerating neurons.
6‐[18F]Fluoro‐L‐DOPA probes dopamine turnover rates in central dopaminergic structures
Authors: Barrio J. R. , Huang S. C. , Melega W. P. , Yu D. C. , Hoffman J. M. , Schneider J. S. , Satyamurthy N. , Mazziotta J. C. , Phelps M. E. 
Published: January 2005

Abstract6‐[18F]Fluoro‐L‐DOPA (FDOPA) cerebral kinetics and metabolism were correlated in normal primates (Macaca nemestrina) and primates with 1‐methyl‐4‐phenyl‐l,2,3,6‐tetrahydropyridine (MPTP) induced unilateral Parkinsonism. Application of a tracer kinetic model to positron emission tomography (PET) data indicated that the model allows reliable estimation of FDOPA blood brain barrier transport, decarboxylation and release of stored 6‐[18F]fluorodopamine (FDA) radioactivity in normal striatum (k4 = 0.005/min, turnover half‐time ≥ 2 hr), in agreement with biochemical data. PET scans of MPTP treated monkeys revealed 40‐50% reduction in total striatal activity in comparison with pre‐MPTP scans. Monkey brain biochemical analysis revealed that the reduction in activity was mainly due to a decrease in FDA and its metabolites, 6[18F]fluorohomovanillic (FHVA) and 6‐[18F]fluoro‐3, 4‐dihydroxyphenylacetic acid (FDOPAC). The remaining activity in tissue was 3‐0‐methyl‐6‐[18F]fluoro‐L‐DOPA (3‐OMFD) of peripheral origin. The (FHVA + FDOPAc)/FDA ratio was 1:2 in normal putamen and ≥6:1 in the lesioned putamen, indicative of a dramatic increase in turnover of FDA. Both kinetic and biochemical data indicate that FDOPA labels a slow turnover rate pool of dopamine in rat and primate brain. This turnover rate for stored dopamine (DA) is accelerated with dopaminergic cell losses (e.g., MPTP‐induccd Parkinsonism).
Reverse‐phase high‐performance liquid chromatography of nerve growth factor receptor‐like proteins identified with monoclonal antibodies
Authors: Shan D.‐E. , Beck C. E. , Werrbach‐Perez K. , Perez‐Polo J. R. 
Published: January 2005

AbstractHuman neuroblastoma SK‐N‐SH‐SY5Y (SY5Y) and rat pheochromocytoma PC12 cells are model cell lines used in the study of nerve growth factor (NGF) effect. The effects of NGF are initiated by binding to cell surface receptors (NGFR). The amino acid sequence for NGFR has been deduced based on the identification of a single gene for NGFR. However, there are two kinds of NGF binding activities and several reported molecular weights of NGFR. We report here on the demonstration of NGFR‐like proteins from PC12 and SY5Y cells by sequential lectin chromatography, reverse‐phase HPLC, and SDS‐PAGE analysis of immunoprecipitates obtained with NGFR‐specific monoclonal antibodies. For both human and rodent NGFR, there was a tendency for the higher molecular‐ weight species of NGFR‐like proteins to be eluted in more hydrophobic fractions. Also, the expression of different species of NGFR could be modified by treatment with retinoic acid (RA). These results are consistent with the hypothesis that the different molecular species of NGFR may result from the generation of a truncated form of NGFR, the presence of sugar residues on the NGFR protein, dimer formation between NGFR, or the association of NGFR with a receptor‐ associated protein.
Characterization of adult human astrocytes derived from explant culture
Authors: Estes M. L. , Ransohoff R. M. , McMahon J. T. , Jacobs B. S. , Barna B. P. 
Published: January 2005

AbstractFour different human astrocytic cell lines established from either epilepsy surgical specimens or cerebral white matter obtained during thalamotomy for tremor in a patient with multiple sclerosis were characterized using morphologic analysis, ultrastructural attributes, growth characteristics, and immunocytochemical analysis. Immunocytochemical characterization of cultures indicated a mean of 84% of cells contained cytoplasmic glial fibrillary acidic protein (GFAP): to confirm that GFAP (+) cells also proliferated, bromodeoxyuridine (BrdU) uptake was measured in cell line. Our method of simplified explant culture allows establishment of astrocytic cell lines from a variety of pathologic substrates using limited amounts of human material.
Acetylcholine‐induced production of platelet‐activating factor by human fetal brain cells in culture
Authors: Sogos V. , Bussolino F. , Pilia E. , Torelli S. , Gremo F. 
Published: January 2005

AbstractPlatelet‐activating‐factor (PAF) is a potent, biologically active lipid mediator produced by several tissues, including brain. Its role in the central nervous system (CNS) is still unknown, even if its involvement in brain damage and neurotoxicity has been postulated. Its production by neural cells has been demonstrated in different species, but not in man. This paper provides evidence that PAF can be produced by human fetal neurons and/or glial cells in culture. Its synthesis dramatically increased upon stimulation with acetylcholine (ACh), and it was significantly lowered by the cholinergic receptor antagonist atropine. Almost no PAF was detected in the incubation medium, which indicated no release of PAF from cultured cells. Characterization of the cells in culture with specific monoclonal antibodies excluded the presence of endothelial cells or macrophages, which also produce PAF.
Folding and function of the myelin proteins from primary sequence data
Authors: Inouye H. , Kirschner D. A. 
Published: January 2005

AbstractTo explain how the myelin proteins are involved in the organization and function of the myelin sheath requires knowing their molecular structures. Except for P2 basic protein of PNS myelin, however, their structures are not yet known. As an aid to predicting their molecular folding and possible functions, we have developed a FORTRAN program to analyze the primary sequence data for proteins, and have applied this to the myelin proteins in particular, In this program, propensities for the secondary structure conformations as well as physical‐chemical parameters are assigned to the amino acids and the pattern of these parameters is examined by calculating their average values, autocorrelation functions and Fourier transforms. To compare two proteins, their sequences are aligned using a unitary scoring matrix, and homologies are searched by plotting a two‐dimensional map of the correlation coefficients.Comparison of the corresponding myelin basic proteins (MBP) and P0 glycoproteins (P0) for rodent and shark showed that the conserved residues included most of the amino acids which were predicted to form the α or β conformations, while the altered residues were mainly in the hydrophilic and turn or coil regions. In both rodent and shark the putative extracellular domain of P0 glycoprotein displayed consecutive peaks of β propensity similar to that for the immunoglobulins, while the cytoplasmic domain showed α‐β‐α folding. To trace the immunoglobulin fold along the P0 sequence, we compared the β propensity curve of P0 with that of the immunoglobulin M603, whose three‐dimensional structure has been determined. We propose that the flat β‐sheets of P0 are orientated parallel to the membrane surface to facilitate their homotypic interaction in the extracellular space. An extra β‐fold in the extracellular domain of shark P0 compared with rodent P0 was found, and this may result in a greater attraction between the apposed extracellular surfaces and may account for a smaller extracellular space as measured by x‐ray diffraction. A computer search of the myelin protein sequences for functional motifs revealed sites for N‐glycosylation, phosphorylation, nucleotide binding, and certain enzyme activities. We note especially that there are potential nucleotide binding sites in proteolipid protein (PLP), MBP and 2′,3′‐cyclic nucleotide 3′‐phosphodiesterase (CNP). This is consistent with the experimental observations that PLP acts like an ionophore or proton channel when reconstituted into planar lipid bilayers, MBP binds GTP, and CNP catalyzes in vitro the hydrolysis of 2′,3′ nucleotides into corresponding 2′‐nucleotides.
Expression of plasmolipin in oligodendrocytes
Authors: Fischer Itzhak , Cochary E. F. , Konola J. T. , Romano‐Clarke G. 
Published: January 2005

AbstractPlasmolipin is a plasma membrane proteolipid which has recently been described as a component of myelin (Cochary et al.: Journal of Neurochemistry 55:602–610, 1990). The present study reports the expression and localization of plasmolipin in primary glial cultures and secondary oligooendrocyte cultures. Double‐label immunofluoresccnce showed that plasmolipin was expressed by galactoccrebroside (GC)‐positive oligodendrocytes, but was absent from astrocytes, characterized by their positive staining for glial fibrillary acidic protein (GFAP). At 1 week in culture plasmolipin staining was relatively weak in the cell body of some of the GC‐positive cells. During the following 3 weeks in culture plasmolipin staining of Oligodendrocytes gradually increased and was present in the cell body, its plasma membrane, and all the processes. However, the plasmclipin antibodies did not stain regions of the flat membrane sheets. Western blot analysis of homogenatcs from primary glial cultures showed that plasmolipin levels gradually increased during the first 5 weeks in culture. We conclude that the presence of plasmolipin in myelin is a result of its expression by Oligodendrocytes.
1,25‐Dihydroxyvitamin D3 is a potent inducer of nerve growth factor synthesis
Authors: Wion D. , Macgrogan D. , Neveu I. , Jehan F. , Houlgatte R. , Brachet P. 
Published: January 2005

Abstract1,25‐dihydroxyvitamin D3(1,25‐(OH)2D3), a metabolically active form of vitamin D, is shown to increase in a dose‐dependent manner the cellular pool of NGF mRNA in murine L‐929 fibroblasts cultured in a serum‐free medium. This effect can be detected as early as 3 hours after 1,25‐(OH)2D3 addition and persists for at least 28 hours. It is accompanied by an enhancement of the amount of NGF‐protein secreted in the culture medium. Since the proto‐oncogene c‐fos appears involved in the regulation of the NGF gene (Mocchetti et al.: Proceedings of the National Academy of Sciences of the United States of America 86:3871‐895, 1989; Hengerer et at: Proceedings of the National Academy of Sciences of the United States of America87:3899‐3903, 1990), the effect of 1,25‐(OH)2D3on c‐fos expression was analysed and compared to that elicited by other inducers of the NGF gene, serum (Wion et al: FEBS‐ Letters 189:37‐41, 1985) and phorbol 12‐myristate 13‐acetate (PMA) (Wion et al: FEBS Letters 262:42‐44, 1990). Addition of serum or PMA to L‐929 cells was; rapidly followed by a transient activation of the c‐fos gene. In contrast,c‐fos transcripts remained undetected in the presence of 1,25‐(OH)3D3 The failure to find any evidence of c‐fos expression suggests that 1,25‐(OH)2D3could enhance the pool of NGF mRNA by a mechanism independent of the c‐fos pathway. The effective concentration range, as low as 10−10 M raises the possibility that 1,25‐(OH)2D3 could represent one of the serum factors that might contribute to the regulation of the NGF gene in vitro and possibly in vivo. This suggests a possible therapeutic interest for 1,25‐(OH)2D3in neurodegenerative diseases.
Co‐activation of insulin‐like growth factor‐I receptors and protein kinase C results in parasympathetic neuronal survival
Authors: Crouch M. F. , Hendry I. A. 
Published: January 2005

AbstractWe have studied the interaction between several growth factors to promote parasympathetic neuronal survival. Neither insulin nor insulin‐like growth factor‐I (IGF‐I) had any effect on the survival of embryonic day 8 chick ciliary neurons in culture. Similarly, the protein kinase C activator phorbol dibutyrate (PdBu) had only a minor survival‐promoting activity. In combination with PdBu, however, IGF‐I or insulin, at concentrations sufficient to act through the IGF‐I receptor, were highly synergistic. In a similar fashion, acidic fibroblast growth factor (aFGF)‐induced neuronal survival was greatly enhanced by PdBu, as well as by insulin or IGF‐I. When added alone, aFGF‐induced cell survival required the presence of 1% serum. However, addition of aFGF, IGF‐I, or insulin with PdBu under serum‐free conditions replaced the serum requirement. That is, these agonist combinations could apparently induce the second messenger requirement for ciliary neuronal survival. Therefore, IGF‐I must now be included in the list of candidate molecules responsible for directing parasympathetic nerve formation. The synergy between agonists observed in these experiments highlights the possibility that combinations of growth factors, rather than sole molecules, may dictate parasympathetic nervous system development in vivo.