bluuebunny/crossref_metadata_2025 huggingface dataset:
Expression of myelin protein genes in quaking mouse brain
Authors: Konat G. , Trojanowska M. , Gantt G. , Hogan E. L.
Published: January 2005

The expression of myelin proteins in actively myelinating quaking and control brains was studied. RNA was extracted from the brains of 18‐ and 27‐day‐old mice and analyzed by northern blot using cDNA probes for proteolipid protein (PLP), basic protein (BP), and myelin‐associated glycoprotein (MAG). Two PLP transcripts of 3.2 and 2.4 kb (kilobase) were found, whereas PB and MAG probes hybridized to single regions of 2.2 and 2.5 kb, respectively. No abnormality in the transcript pattern was detectable in the quaking brain at either 18 or 27 days of age. Over this 9‐day period the level of PLP and BP message in the control brain decreased by approximately 10%, whereas the level of MAG message decreased by approximately 50%. In the 18‐day‐old quaking brain the expression of PLP and BP was severely reduced amounting to one‐third and one‐half of the control values, respectively. The reduction at the age of 27 days was less. On the other hand, the quaking brain produced more MAG mRNA amounting to 1.6‐ and 3.2‐fold control on the 18th and 27th day. The results indicate a reduced expression of the PLP and BP genes and a developmental delay in the mutant, whereas the genetic expression of MAG is enhanced and appears to be progressively dysregulated.
Fetal cortical astrocytes migrate from cortical homografts throughout the host brain and over the glia limitans
Authors: Goldberg W. J. , Bernstein J. J.
Published: January 2005

The cerebral cortices from 14‐day gestation rat embryos were prelabeled with Phaseolus vulgaris leucoagglutin (PHAL) and then homografted into freshly made implantation pockets in the host cerebral cortex. Animals were sacrificed at 30 and 60 days postimplantation (DPI). Paraffin sections were double labeled for the presence of glial fibrillary acidic protein (GFAP), a specific marker for astrocytes, and PHAL, utilized as a marker for transplant‐derived cells. Transplant‐derived astrocytes were found on the glia limitans along the entire circumference of the brain, in the hippocampal commissure, corpus callosum, internal capsule, entopeduncular nucleus, habenular commissure, brachium of the superior colliculus, optic tract, optic chiasm, and sensory root of the trigeminal nerve. Transplanted astrocytes entered the spaces of Virchow‐Robin, and migrated along parenchymal blood vessels and between the ependymal and subependymal layers of the third and lateral ventricles. The presence of basal lamina or parallel nerve fiber bundles was a common factor for these migration routes.
Direct microculture enzyme‐linked immunosorbent assay for studying neural cells: Oligodendrocytes
Authors: Gard A. L. , Warrington A. E. , Pfeiffer S. E.
Published: January 2005

Oligodendrocyte development has been studied in a standardized primary microculture system initiated from day 20‐21 fetal rat brain using a solid‐phase enzyme‐linked immunosorbent assay (ELISA) carried out directly on fixed cells (direct microculture ELISA). A highly reproducible dissociation procedure is described that allows careful control of the number of cells seeded per culture. At a seeding density of 1 × 105 cells/culture, up to 250 oligodendrocyte‐generating microcultures consisting of 10‐12% oligodendrocytes can be prepared from a single fetal rat brain, thereby permitting the simultaneous assay of multiple developmental parameters in sibling cultures. The validity of this method for quantifying myelinogenesis was established by comparing the results obtained by direct microculture ELISA with immunocytochemical counting of cells in parallel cultures. As few as 200 oligodendrocytes could be detected using a biotinylated anti‐Ig and an avidin‐urease coniugate detection system; CNP immunoreactivity measured by ELISA was linearly proportional to the number of immunolabeled cells between 6 and 34 days in culture; the developmental time courses of 2′,3′‐cyclic nucleotide 3′‐phosphohydrolase (CNP) and myelin basic protein (MBP) expression determined by the two methods were very similar. Finally, cell suspensions were seeded at increasing dilution to determine the number of cells required to generate cultures that tested positive for oligodendrocytes by ELISA. As few as 9,000 cells were sufficient, predicting a minimum of 8,000 oligoprogenitors per 20‐21 day fetal rat brain. The application of direct microculture ELISA for studying oligodendrocyte population size and myelinogenesis is discussed.
Alterations of the posttranslational processing of a lysosomal enzyme in C6 glioma cells
Authors: Snyder D. S. , Whitaker John N.
Published: January 2005

Cathepsin D was assessed in C6 glioma cells grown in medium with an intermediate‐ or low‐percent composition of serum. The amount, form, and subcellular location of cathepsin D differed after treatment with cyanate or monensin in cells grown in a low‐serum, growth‐factor‐supplemented medium. Immunoblotting showed that cathepsin D in the lysosomal fraction of the C6 cell line had a molecular weight (Mr) of 42 kD, whereas that in the microsomal fraction had Mr's of 42, 47, and 78 kD. After treatment for 1 to 16 hr with 4 mmol/L cyanate and subcellular fractionation, the molecular weight of lysosomal cathepsin D was the same in treated and untreated cells, but more enzyme was found in lysosomes of treated cells at 8 and 16 hr. In the microsomal fraction, the amounts of both the 42 and 47 kD forms were increased after 1 to 16 hr of treatment. When exposed to 20 mmol/L cyanate, C6 cells remained viable, but compared with untreated cells, they showed 25% less lysosomal cathepsin D, with increased amounts found in the microsomal fraction. The 78 kD protein detected by immunoblotting was present in both the lysosomal and microsomal fractions but was predominant in the latter. The apparent molecular weight of this protein was the same after cyanate but differed with monensin, where Mr's of 39, 42, and 73 kD were found. Monensin‐treated cells had less lysosomal cathepsin D and relatively more microsomal enzyme. The differing molecular weights of cathepsin D from cyanate‐ and monensin‐treated cells suggest that their inhibitions occur at different processing loci in distal elements of the Golgi stacks. The differences in the pI of cathepsin D and the number of its forms from cyanate‐ and monensin‐treated cells are also consistent with interference in the late stages of glycoprotein maturation.

In this paper we show that the amount, molecular form, and consequent intracellular location of cathepsin D in cells of the C6 line can be affected by agents that selectively disrupt stages in Golgi‐related protein modification and transport.
Number of olfactory marker protein‐containing receptor cells is influenced by developmental stage of the olfactory bulb
Authors: Chuah M. I. , Au C.
Published: January 2005

In vitro studies on E15 rat embryos have shown that the number of receptor neurons containing olfactory marker protein (OMP) is markedly increased when olfactory mucosa (OM) is cultured in direct contact with the presumptive olfactory bulb (POB). This facilitatory influence is tissue‐specific; that is, it is absent when the POB is substituted with other nervous or non‐nervous tissues. In the present quantitative immunohistochemical study, we show that the enhancing influence of the POB is also a stage‐specific phenomenon. The number of OMP‐containing receptor cells is greatest when E15 OM is cultured with POB of the same age. If the POB was taken from a less mature (E13) or from a more mature (E17) embryo, the number of OMP‐containing cells was greatly reduced.
Two types of focal accumulations of acetylcholinesterase appear in noninnervated regenerating skeletal muscles of the rat
Authors: Sketelj J. , Črne N. , Brzin M.
Published: January 2005

Muscle fibers in the soleus muscle of the rat, injured by bupivacaine and free autografting, were allowed to regenerate within their old basal laminae. Histochemical and cytochemical analysis of newly synthesized acetylcholinesterase (AChE) revealed that two kinds of focal accumulations of AChE gets concentrated at the sites of the former motor endplates. Accumulation of AChE starts in places where a tight contact between the remnants of the old junctional basal lamina and the budding surface of the myotube engulf the extracellular material. Appearance of these AChE accumulations can be prevented by papain treatment of the soleus muscle before autografting but not by predenervating it for 1 month. Focalization of AChE is probably induced by a component of the junctional basal lamina, possibly a protein, the existence of which is not dependent upon continuous presence of the motor nerve and may be produced by the muscle. This view is corroborated by the fact that an additional kind of AChE accumulation appeared in regenerating muscles in regions remote from the sites where motor endplates were located in the muscles of origin. Although differing in localization, size, and appearance, both kinds of AChE accumulations ultrastructurally resemble the postsynaptic specialization of the motor endplate: they consist of tubelike sarcolemmal invaginations containing AChE. The extrajunctional AChE accumulations seem to arise spontaneously and are usually located more than 750 μm away from the junctional ones as if some local inhibitory mechanism prevents their formation in the immediate vicinity.
Serum contains inducers and repressors of oligodendrocyte differentiation
Authors: Bologa L. , Cole R. , Chiappelli F. , Saneto R. P. , De Vellis Jean
Published: January 2005

An important stage in oligodendrocyte development is the expression of galactocerebroside (GC), the major glycolipid in myelin. Although oligodendrocyte cell lineage and differentiation in vitro have been the object of many studies, to date there is sparse information on the regulation of GC expression in oligodendrocytes already committed to be positive for GC. We report here that GC expression in these cells is controlled by three serum factors. Two of these, possibly a lipoprotein and a mucoprotein, increase GC levels, whereas the third, probably a glycoprotein, exerts an inhibitory effect. The developmental increase of GC in postnatal rat brain cerebral cultures and its induction by serum factors are reversible phenomena. The isolation of the GC‐regulatory factors would allow experimental manipulation of impaired GC expression by differentiated oligodendrocytes.
Decreased levels of nerve growth factor receptor on dexamethasone‐treated PC12 cells
Authors: Tocco Maria D. , Contreras M. L. , Koizumi S. , Dickens G. , Guroff G.
Published: January 2005

Treatment of PC12 cells with dexamethasone leads, in a period of days, to a 60% decrease in the binding of (125I)nerve growth factor. The decrease was maximal after 3 days of treatment with 1μM dexamethasone, but some decrease was seen after 6 hr and at concentrations as low as 10 nM. The effect was specific for the glucocorticosteroids. Scatchard plots confirmed the overall loss of nerve growth factor binding, and studies with trypsin digestion and Triton X‐100 extraction indicated that the decrease in binding was largely due to a decrease in the number of low‐affinity receptors. Nerve growth factor‐induced changes, such as the induction of ornithine decarboxylase and the generation of neurites, were inhibited, but only minimally, in dexamethasone‐treated cells.
Expression and localization of the fibronectin receptor in the mouse nervous system
Authors: Pesheva P. , Juliano R. L. , Schachner Melitta
Published: January 2005

Cell surface receptors for extracellular matrix components have recently been characterized as integral membrane complexes with common features in their structural and functional properties. We have investigated the expression of the mammalian fibronectin receptor in the mouse nervous system using immunocytological and immunochemical methods. The fibronection receptor was detectable on immature oligodendrocytes and immature and mature astrocytes in culture, while central nervous system neurons did not reveal detectable levels of fibronectin receptor at the developmental stages studied. In the peripheral nervous system both glia and neurons were found to express the fibronectin receptor. The receptor complex in both peripheral and central nervous system has an apparent molecular weight of approximately 140 kD under reducing conditions and resolves into two or three distinct protein bands under nonreducing conditions. The fibronectin receptor expresses the L2/HNK‐1 epitope that is characteristic of several adhesion molecules, including L1, N‐CAM, the myelin‐associated glycoprotein, and J1 and thus is another member of the L2/HNK‐1 family of adhesion molecules. The L2/HNK‐1 carbohydrate epitope is expressed differently and independently of the fibronection receptor protein backbone in that it is detectable in neonatal brain but not in adult brain. Our observations attribute a functional role to the fibronectin receptor and its L2/HNK‐1 carbohydrate epitope during development and maintenance of cell interactions in the central and peripheral nervous systems.
Dolichol‐linked oligosaccharide and glycoprotein biosyntheses in glial cells in primary culture: Development and enzymatic correlates
Authors: Ishii Satoshi , Volpe Joseph J.
Published: January 2005

Primary cultures of cerebral glia derived from neonatal rat brain were utilized to determine (1) the developmental changes of dolichol‐linked oligosaccharide and N‐linked glycoprotein biosyntheses, (2) the enzymatic correlates of these developmental changes, and (3) the temporal relations between the biosyntheses of dolichol‐oligosaccharide and N‐linked glycoproteins and the biochemical expression of astrocytic and oligodendroglial differentiation. Marked, parallel developmental increases in the rates of incorporation of [3H]glucosamine into both dolichol‐oligosaccharide and glycoprotein were observed, with maximal rates achieved after 9–10 days in culture and little change occurring over the next 10 days in culture. Concerning the enzymatic correlates, dolichol kinase exhibited a moderate developmental increase with a maximum at 5 days in culture, whereas the activities of the three critical enzymes that utilize dolichyl phosphate in the synthesis of the dolichol‐linked oligosaccharide, i.e., N‐acetylglucosaminylphosphotransferase (GlcNAc‐1‐P transferase), mannosylphosphoryldolichol (Man‐P‐Dol) synthase, and glucosylphosphoryldolichol (Glc‐P‐Dol) synthase, reached maxima after 6–9 days in culture. Both the activity of Man‐P‐Dol synthase in vitro and the rate of formation of its product, Man‐P‐Dol, in intact cells were shown to correlate closely with the rates of oligosaccharide and glycoprotein biosyntheses. An important regulatory role for Man‐P‐Dol synthase and its product, Man‐P‐Dol, was suggested further by the demonstration of a maturation‐dependent activation by Man‐P‐Dol of GlcNAc‐1‐P transferase, the first committed step in the pathway. Two enzymatic markers of astrocytic (glutamine synthetase) and oligodendroglial (2′,3′‐cyclic nucleotide 3′‐phosphohydrolase) differentiation exhibited marked developmental increases in activity with onset at the time of attainment of peak rates of dolichololigosaccharide and glycoprotein biosyntheses. Importance of the latter processes for glial differentiation is suggested.
Chromosomal locations of genes encoding 2′,3′ cyclic nucleotide 3′‐phosphodiesterase and glial fibrillary acidic protein in the mouse
Authors: Bernier L. , Colman D. R. , D'Eustachio P.
Published: January 2005

Cyclic nucleotide phosphodiesterase (CNP) and glial fibrillary acidic protein (GFAP) are useful markers of myelin and astroglia, respectively. Two proteins with CNP activity are known to exist in brain and lymphoid tissues. They appear to be the products of several distinct but related messenger ribonucleic acid (mRNA) species. GFAP is a single protein encoded by a single mRNA. We have localized the GFAP gene to distal chromosome 11 in the mouse. There are two genetic loci identified by CNP probes, one is closely linked to the GFAP gene, and the other maps to chromosome 3.
Role of antibodies in T cell‐mediated experimental allergic encephalomyelitis
Authors: Hashim G. A. , Day E. D.
Published: January 2005

The role of the humoral phase of the immune response in development of T cell‐mediated experimental allergic encephalomyelitis (EAE) had not been clearly defined previously even though studies of the myelin basic protein (MBP) molecule had demonstrated the presence not only of T cell but also B cell epitopes capable of inducing cell‐mediated immunity and antibody formation. Particularly relevant to this report are the immunological expressions of the region which induces EAE in the Lewis rat. The development of primary demyelination in Lewis rats is preceded by a cell‐mediated immune response as well as antibody formation, both of which are highly specific to the encephalitogenic 14‐residue peptide that defines the 69–84 region of the parent MBP. Our results are consistent with the dogma that EAE is a T cell‐mediated disease, but they also clearly demonstrate an important role for specific antibodies in the development of these T cells responsible for demyelination. The antibody response, which may be heteroclitic, is necessary for T cells to develop into an effector T cell subset. Without this B cell response the subsequent T cell response does not lead to demyelination. In this report we shall discuss these findings and further show that the T cell and B cell epitopes, which are located within the 14‐residue sequence, are physically separated and dependent upon the form of synthetic peptides known to induce T cell‐mediated and/or humoral immunity.

Although neither of the two kinds of epitopes when injected alone induced primary demyelination, when administered together or separately they induced EAE similar to that induced by the parent 14‐residue peptide. Moreover, antibody against the specific B cell epitope could substitute for the epitope itself in the development of disease. The T cell epitope by itself failed to induce any detectable antibody. Unlike other reported anti‐MBP antibodies, the antibody against the specific MBP epitope reported here is thus required for T cell activation even though it does not participate directly in the development of primary demyelination in EAE.
Cell adhesion and neurite extension in response to two proteolytic fragments of laminin
Authors: Rogers S. L. , Palm S. L. , Letourneau P. C. , Hanlon K. , McCarthy J. B. , Furcht L. T.
Published: January 2005

Studies from several laboratories have suggested that laminin contains at least two domains that selectively mediate cell type‐specific behavior. In this study, two proteolytic fragments of laminin are evaluated for their ability to interact with three different populations of embryonic chicken cells. A 600 kDa thrombin fragment, derived from the central portion of the laminin molecule, supports attachment of dorsal root ganglion (DRG), spinal cord (SC), and heart cells. Neurons from both DRGs and SCs extend neurites in response to this fragment. Quantitatively, both cell adhesion and neurite extension on the 600 kDa fragment are comparable to these responses to intact laminin. A 440 kDa chymotrypsin fragment, derived from either intact laminin or the 600 kDa fragment, does not support equivalent responses. Fewer DRG cells attach to this fragment and neurites are shorter than on the 600 kDa fragment. Heart and SC cell attachment is also reduced in comparison with activity of the 600 kDa fragment, and SC neurites do not form on the 440 kDa fragment. These results suggest that there are at least two cell binding domains in the laminin molecule, one with which a variety of cell types can interact and another that may mediate more restricted cellular responses. The latter site appears to be relatively inactive for SC and heart cell adhesion but supports limited attachment and neurite extension by DRG neurons.
Differentiating astroglia in nervous tissue histogenesis/regeneration: Studies in a model system of regenerating peripheral nerve
Authors: Kalderon N.
Published: January 2005

The role of astroglia in nervous system histogenesis/regeneration (morphogenesis) was studied as a function of cell age. The effect of inoculated astroglia at different cell maturation stages on axonal growth was examined in a peripheral nerve regenerating model system. This model system consists of rat sciatic nerve stumps that regenerate through an empty silicone chamber (Lundborg et al.: Journal of Neuropathology and Experimental Neurology 41:412–422, 1982). Rat astroglial cell populations grown in cell culture were suspended either in a liquid (physiological solution) or in a solid (isotonic collagen gel) medium and inoculated within the silicone chamber at the time of surgery. Immature and mature cell populations, at ages corresponding to 9–69 postnatal days (P9–P69), were inoculated, and their effect on neural growth was analyzed by histological, immunocytochemical, and ultrastructural techniques, 2–6 weeks later.

Astroglial cells differentially modulated the process of nerve regeneration, an effect that is a function of the cells' developmental stage. P19 astroglia and older cells inhibited the regeneration process, encapsulating the axons in a glia‐limitans‐like structure. Immature astrocytes (P9) did not seem to interfere with the regeneration process; nerve outgrowth in their presence resembled and was comparable to the ones obtained in the presence of inoculated Schwann cells. The differential effects of the developing astroglia were not significantly changed by the inoculation media, e.g., liquid or solid, and were already pronounced 2 weeks after neural transection.

It is suggested by the results of the study that the role and function of astroglia in nervous system morphogenesis are changing with cell differentiation. Adult astrocytes seem to downregulate axonal growth; presumably, their function is to confine the neurites within designated structural and functional boundaries.
A normal human brain cell aggregate model for neurobiological studies
Authors: Pulliam L. , Berens M. E. , Rosenblum M. L.
Published: January 2005

A new in vitro model of normal human brain has been developed in which fetal human brain cells form three‐dimensional aggregates that can be maintained for up to 60 days in culture. Cells appear fully differentiated at the time of initiation in culture; the predominant cells identified were astrocytes, neurons, and oligodendrocytes with myelin, with occasional ependymal cells and macrophages. The specific arrangement and numbers of neural cells within aggregates differed among brain specimens. Cell kinetics studies detected DNA synthesis throughout the culture interval. Aggregates cocultured with a human malignant glioma cell line (U251‐MG) were progressively invaded by tumor cells. In aggregates infected with human cytomegalovirus (CMV), intracellular viral replication and morphologic changes characteristic of human brain infection with this pathogen were seen. This model of brain aggregates should prove valuable for multidisciplinary studies in human neurobiology, particularly in the fields of developmental neurobiology, neurooncogenesis, tumor cell invasion, and species‐specific viral infection of the central nervous system.
Monoclonal antibody that inhibits biological activity of a mammalian ciliary neurotrophic factor
Authors: Watters D. , Belford D. , Hill C. , Hendry I.
Published: January 2005

Four monoclonal antibodies to a purified ciliary neurotrophic factor have been produced by a combination of in vivo and in vitro immunization of mouse lymphocytes. Three of the antibodies—3D12, an 1gA subtype, and 5G9 and 2B11, both IgM subtypes—bound to the single band on immunoblots of the purified factor. Of the four clones only one, 3D12, produced antibodies able to inhibit the biological activity of the neurotrophic factor in promoting the survival of ciliary neurones in dissociated culture.
Is part of the molecular basis of the perineurial barrier function the lack of endogenous carbohydrate‐binding proteins?
Authors: Bardosi A. , Dimitri T. , Behrends T. , Autschbach D. , Gabius H.‐J.
Published: January 2005

The sugar part of cellular glycoconjugates and specific endogenous sugar receptors, i.e., lectins, can establish a system of biological recognition based on proteincarbohydrate interactions. An assortment of labelled (neo)glycoproteins, carrying different types of sugar moieties, is synthesized to localize respective sugar receptors. With these tools, the histochemical patterns of endogenous carbohydrate‐binding receptors of the epi‐, peri‐, and endoneurium were analyzed in human sural and accessory nerves and in swine sciatic nerve. This approach is complementary to the application of plant lectins, focusing on endogenous carbohydrate‐binding proteins (lectins). In contrast to the epi‐ and endoneurium, which bound certain types of carbohydrates, such endogenous sugar receptors were histochemically not detectable in the perineurial cells. Moreover, no histochemical reaction was present in the “connective tissue septa” localized in the endoneurium in which the endoneurial vessels were embedded. This common property supplies evidence that these septa are composed of perineurial cells. They may represent a barrier in addition to the capillary endothelium. Our observations suggest histogenetical differences between the cell populations of epi‐and endoneurium vs. perineurium. This significant difference in the ability to bind carbohydrate residues, conjugated to a carrier protein, is contradictory to the assumption that perineurial cells and fibroblasts are functional variants of the same cell type.

The histochemical patterns of endogenous carbohydrate‐binding receptors found in human and swine nerves were similar but not identical, with exception of the perineurium, reflecting phylogenetic differences in the expression of sugar‐binding proteins. The absence of specific sugar receptors in perineurial cells, however, seems to be a more general phenomenon. It can lead to a profoundly limited recognitive capacity of these cells within a sugar‐code system of biological information, possibly contributing to the molecular basis of the barrier function.
Expression of carbonic anhydrase II gene in early brain cells as revealed by in situ hybridization and immunohistochemistry
Authors: De Vitry F. , Gomes D. , Rataboul P. , Dumas S. , Hillion J. , Catelon J. , Delaunoy J.‐P. , Tixier‐Vidal A. , Dupouey P.
Published: January 2005

A mouse carbonic anhydrase (CA II) complementary(c) DNA probe was used for in situ hybridization on mouse brain cultured cells in order to follow CA II gene expression during brain development. An improved method was established using biotinated probes that resulted in a high sensitivity and an absence of background; this method could be combined with immunohistochemistry. Hypothalamic cells of embryonic day (ED) 12–14 mice were cultured for various periods. Chronologic appearance of CA II messenger(m)RNA and protein was studied. The CA II gene transcripts are detectable as early as ED 12–13, although the protein they encode is not detectable until ED 17–18. Gene expression is restricted to 0.1% of the total population. Northern blot analysis confirmed the presence of CA II transcripts in embryonic hypothalamus. At postnatal stage, the majority of glial cells express both the CA II mRNA and the protein. Our results favour the early appearance of a glial lineage in a precise area of the developing CNS. The precocity of CA II gene transcription makes in situ hybridization an invaluable approach in defining the onset of nerve cell lineages during embryonic development.
Stimulus‐coupled taurine efflux from cerebellar neuronal cultures: On the roles of Ca++ and Na+
Authors: Philibert R. A. , Rogers K. L. , Dutton G. R.
Published: January 2005

Primary cultures of cerebellar neurons obtained from 7–9‐day‐old rats and grown 7–9 days in vitro (DIV) were used to study the effects of Na+ and Ca++ on K+‐evoked taurine release. These cultures, made up largely of granule neurons (90%) and inhibitory interneurons (5–7%), produced a dose‐dependent, depolarization‐evoked taurine release that was Ca++‐dependent at 40 mM K+, and Ca++‐independent at K+ concentrations above 40 mM. The dihydropyridine Ca++ channel agonist BAY K 8644 (1 μM) augmented 30 mM K+‐evoked release, while the antagonist nifedipine (5 μM) abolished both the BAY K 8644‐ and K+‐enhanced release. Depolarization with the Na+ channel agonist veratridine (50 μM) stimulated taurine efflux, which was completely blocked by pretreatment with tetrodotoxin (2 μM). However, 50 mM K+‐evoked taurine release was not affected by tetrodotoxin pretreatment. Substitution of choline Cl for NaCl partially antagonized 50 mM K+‐evoked release, and by itself, the Na+ ionophore monensin (50 μM) stimulated release. These results suggest that both K+‐evoked and basal taurine release from primary cerebellar neuronal cultures are sensitive to the levels of both intracellular and extracellular Na+ and Ca++ In contrast to previous findings using cerebellar astrocytes, neuronal L‐type Ca++ channels, but not voltage‐dependent Na+ channels, also appear to be necessary. The implications of these results on taurine's status as a putative neurotransmitter are discussed.
Brain neocortex influence on the mononuclear phagocyte system
Authors: Neveu P. J. , Barnéoud P. , Georgiades O. , Vitiello S. , Vincendeau P. , Le Moal M.
Published: January 2005

The cerebral neocortex is known to modulate asymmetrically certain components of the immune system. It was previously shown that large ablation of the left cortex reduces B and T cell‐mediated responses, whereas symmetrical right lesions enhance these responses. We have studied the immunomodulatory role of the brain cortex on the mononuclear phagocytic system. Resident and BCG‐activated macrophages were investigated in female C3H/He mice at 8–10 weeks after right or left cortical ablation. After an intraperitoneal injection of BCG, the number of peritoneal macrophages was found to be lower in both right‐ and left‐lesioned mice, the difference being stronger and more significant in left‐lesioned animals than in sham‐operated controls. Furthermore, the oxidative metabolism as assessed by chemiluminescence was depressed only in left‐lesioned mice. On the other hand, cortical lesions were shown to have no effect on either the number or the endocytic activity of resident peritoneal macrophages. The possible implication of the brain neocortex on infectious diseases was assayed by using the Trypasonoma musculi model, in which macrophages are known to be effective in parasite eradication. Although the number of peritoneal macrophages was significantly depressed after left cortical lesions 11 days after T. musculi inoculation, the course of the infection was not modified significantly. Our results argue in favor of brain neocortex modulation of the mononuclear system.
Paradoxical and subtype‐specific effects of opiate antagonists on the expression of opioid receptors in rat brain cultures
Authors: Barg J. , Levy R. , Simantov R.
Published: January 2005

Cultures of aggregating fetal rat brain cells express μ, δ, and k opioid receptors. The potent and longlasting opioid antagonist naltrexone was used to investigate whether different regulatory mechanisms are involved in the expression of the three receptor subtypes. In cultures treated for seven days, naltrexone increased dose‐dependently the binding of 3H‐diprenorphine to the three receptor subtypes, with the μ sites being affected at a lower concentration than the other two; A Scatchard analysis indicated that this receptor up‐regulation was obtained by an increase in the BMax, with no significant change in the affinity of the ligand to the receptors. In contrast to these effects in cultures treated for 7 days, it was surprising to find that a 48 hr treatment with naltrexone had an apparent converse and subtypespecific influence; the antagonist decreased significantly the binding of 3H‐diprenorphine to both μ and δ receptors but had no effect on k sites. Two other opioid antagonists, naloxone and levallorphan, had a similar effect. Further analysis of naltrexone's mode of action was obtained by studying its effect on the adenylate cyclase activity. Of several inducers of this enzyme, the β‐adrenergic inducer isoproterenol gave the highest increase in cyclic AMP. Naltrexone had no significant effect on the basal adenylate cyclase activity but it altered the pattern of cyclic AMP formation in isoproterenol‐stimulated cultures. Overall, the results indicate that in addition to its classic antagonistic activity, naltrexone exhibits in fetal brain aggregates some properties associated with opiate agonists.
Age‐related modification of enzyme activities in synaptosomes isolated from rat cerebral cortex
Authors: Curti D. , Benzi G.
Published: January 2005

In synaptosomes from rat cerebral cortex, the potential catalytic activity of some enzymes related to energy metabolism—namely, phosphofructokinase and citrate synthase—is not affected by aging. In contrast, the maximum velocity (Vmax) of cytochrome oxidase and of pyruvate dehydrogenase decreases in aged rats. A marked increase is found in the Vmax of glucose 6‐phosphate dehydrogenase in aged rats and could be related to the availability of NADPH for antiperoxidative processes.

Pretreatments of experimental animals with certain drugs were done to investigate the plasticity of enzyme proteins during aging. Papaverine, which acts on macrocirculation, is ineffective, but delta‐yohimbine acting on microcirculation and metabolism and almitrine acting on oxygen availability both could interfere with the potential activity of some enzymes. However, their influence differs with the age of the rats.
Influence of laminin on the responsiveness of early chick embryo neural tube neurons to nerve growth factor
Authors: Heaton M. B.
Published: January 2005

Dissociated neurons from the neural tube containing the trigeminal motor nucleus from early chick embryos were cultured on laminin or collagen‐polyornithine substrates, with and without nerve growth factor (NGF). Control cultures were grown in similar conditions with cytochrome‐C. It was found that neuronal survival was not affected by NGF or cytochrome‐C, but it was enhanced by laminin. The expression of neuritic processes, however, was significantly enhanced in the presence of NGF on both laminin and collagen‐polyornithine surfaces, with the greatest number of neurons producing processes seen in the laminin‐plus‐NGF group. The length of processes was similarly enhanced by laminin and by NGF. Cytochrome‐C did not influence any of these measures. The results indicate that while laminin potentiates the NGF effect on these early neuronal populations, NGF in conjunction with other substrata can have similar, though less dramatic, effects. These results, together with prior evidence of NGF receptors and specific NGF retrograde transport, suggest that this growth factor may play a significant role in the normal ontogeny of early motoneuron populations.
Monoclonal antibody detects embryonic epitope specific for nerve‐derived transferrin
Authors: Festoff B. W. , Munoz P. A. , Patel M. K. N. , Harris M. , Beach R. L.
Published: January 2005

Monoclonal antibodies were generated against transferrin purified from chick embryo extract by fusing spleen cells from BALB/∼c mice immunized against embryonic transferrin, with myeloma cells. Antibodies produced by the selected hybridoma clones were all type IgG. Twelve clones were selected for secretion of antibodies to the embryo extract‐derived transferrin, and three clones were studied extensively. Immunoblotting was used to demonstrate antibody binding to several avian transferrin proteins derived from adult chicken serum, adult chicken peripheral nerves, and ovotransferrin. Screening and detailed epitope analysis were accomplished by solid‐phase immunoassay. The results indicated that two clones, 2G9.1 and 2B11.1, recognized the embryonic and egg antigens in preference to the adult proteins. However, a third clone, 6H2.1, recognized the nerve‐derived transferrin preferentially to both the embryonic and adult serum antigens. None of the clones recognized the serum‐derived transferrin in preference to the other antigens. These results indicate that embryonic epitope(s) are conserved in the nerve‐ but not the serum‐derived transferrin. They also show that the neural antigen has site(s) distinct from the embryonic proteins. No changes in displacement curves were observed after these proteins were digested with neuraminidase, indicating that the epitope differences discovered are not intimately related to sialic acid residues on the various transferrins.
Response of human T lymphocyte lines to myelin basic protein: Association of dominant epitopes with HLA class II restriction molecules
Authors: Chou Y. K. , Vainiene M. , Whitham R. , Bourdette D. , Chou C. H.‐J. , Hashim G. , Offner H. , Vandenbark A. A.
Published: January 2005

In animals, the selection in vitro of T cell lines to myelin basic protein (MBP) can define immunodominant and encephalitogenic epitopes which are preferentially associated with class II major histocompatibility (MHC) molecules. These principles were used to evaluate the specificity and MHC restriction of 14 human MBP‐reactive T cell lines selected from normal individuals and patients with multiple sclerosis (MS) and other neurological diseases (OND). The four normal T cell lines recognized single, separate immunodominant MBP epitopes which were restricted by MHC molecules from the DR or in one case the DP class II locus. In contrast, the MS and OND T cell lines recognized multiple MBP epitopes, each in association with a discrete class II MHC molecule from the DR or DQ locus. Overall, HLA‐DR molecules were used preferentially to associate with epitopes on human MBP, restricting 26/33 responses. As predicted from animal studies, T cells from genetically disparate individuals responded to different immunodominant epitopes on human MBP in association with distinct MHC class II molecules. HLA‐DR2, which is overrepresented in MS patients, possessed an unusual capacity to restrict all eight epitopes identified on MBP in this study. These data provide the first evidence of genetically restricted human T cell recognition of potentially encephalitogenic epitopes of MBP.
Presence of α‐melanocyte‐stimulating hormone‐like immunoreactivity in the innervation of amphibian skeletal muscle
Authors: Oren N. , Micevych P. E. , Letinsky M. S.
Published: January 2005

Amphibian motor nerve terminals are sensitive to a wide variety of peptides, including α‐melanocytestimulating hormone (α‐MSH). We determined the presence and distribution of α‐MSH‐like immunoreactivity (α‐MSHLI) in the innervation of the cutaneous pectoris muscle from bullfrog (Rana catesbeiana) tadpoles and postmetamorphic froglets, and adult frogs (R. catesbeiana and R. pipiens). α‐MSHLI was found in unmyelinated, noncholinergic axons, in motor axons, and in motor nerve terminals. In motor axons, α‐MSHLI was predominantly associated with neurofilaments. The distribution of this form of α‐MSHLI changed during development and seasonally in adult frogs. The possible functional roles of this α‐MSHLI are discussed.
Induction of morphological differentiation of human neuroblastoma cells is accompanied by induction of tissue‐type plasminogen activator
Authors: Neuman T. , Stephens R. W. , Salonen E.‐M. , Timmusk T. , Vaheri A.
Published: January 2005

Human SH‐SY5Y neuroblastoma cells treated with retinoic acid, 12‐O‐tetradecanoyl‐phorbol‐13‐acetate (TPA) or nerve growth factor differentiated morphologically to neuronlike cells with increased amounts of neurofilament protein and mRNA. All three effetors induced an increase in the amount of relative molecular weight (Mr) 70 000 tissue‐type plasminogen activator (t‐PA) and its mRNA, as determined by immunocapture, enzyme activity, and Northern blotting analyses. About 90% of the t‐PA activity was secreted to the culture medium. In contrast, of the three effectors studied, only TPA induced transcription of the proto‐oncogene c‐fos, studied as a control gene responsive to various stimuli, and induced a rapid increase in urokinase‐type PA (u‐PA). Most of the u‐PA activity induced by TPA remained cell‐associated. Because induction of differentiation correlated closely with induction of t‐PA, and not u‐PA, the authors propose that t‐PA may have a functional role in the morphological differentiation of neuronal cells.
GABAA and GABAB receptors are functionally active in the regulation of catecholamine secretion by bovine chromaffin cells
Authors: Castro E. , Oset‐Gasque M. J. , González M. P.
Published: January 2005

GABA stimulates the basal catecholamine release from adrenal bovine chromaffin cells in a calciumdependent manner. This release represents about 70% of that obtained by similar doses of nicotine under similar experimental conditions. This effect is mediated by GABAA receptor sites present in chromaffin cells, since it was mimicked by muscimol and reversed by bicuculline. In addition, GABA, through its GABAA receptors, increases the catecholamine release evoked by submaximal doses of aicotine, but it has no effect on nicotine‐evoked secretion of catecholamines when nicotine was given at maximal doses. These results seem to indicate that both nicotine and GABA release catecholamines from the same intracellular pool. In contrast, baclofen, a GABAB receptor agonist, depressed both basal and nicotineevoked catecholamine release; this result indicates that in addition to GABAA control of catecholamine secretion by chromaffin cells, there is a GABAB control of this function. These results support the existence of a dual regulation of catecholamine secretion by both the GABAA and GABAB receptors in a similar way as that proposed for muscarinic and nicotinic cholinergic receptors.
Isolation, characterization, and application of monoclonal antibodies to rat tyrosine hydroxylase
Authors: Kwan S.‐W. , Patel N. T. , Vulliet P. R. , Hall F. L. , Denney R. M. , Shen R. S. , Westlund K. N. , Abell C. W.
Published: January 2005

Tyrosine hydroxylase (TH, tyrosine 3‐monooxygenase; EC 1.14.16.2) activity in crude extracts of rat pheochromocytoma, rat brain, and bovine adrenal medulla can be immunoprecipitated in an indirect assay by monoclonal antibodies prepared against partially purified rat pheochromocytoma TH. One of tese monoclonal antibodies, TH‐2D8hyphen;2, can be used for immunocytochemical localization of TH in cell bodies, dendrites, and axons in catecholaminergic neurons (e.g., cells in the substantia nigra) of rat brain and in the cell body, neurites, and growth cones of rat pheochromocytoma cells after treatment with nerve growth factor. When linked to Affihyphen;gel 10, this monoclonal antibody can also be used for immunoaffinity purification of rat and bovine TH. These results suggest that THhyphen;2D8hyphen;2 is a valuable reagent with which to investigate the localization, physiological regulation, and function of this important enzyme.
Post‐translational processing in model neuroendocrine systems: Precursors and products that coordinate reproductive activity in Aplysia and Lymnaea
Authors: Nagle G. T. , Painter S. D. , Blankenship J. E.
Published: January 2005

The regulation of egg‐laying behavior in the hermaphroditic marine mollusc Aplysia has been an important model system that has provided insight into the cellular and molecular bases of neuroendocrine function. The advantages of this system derive primarily from the unique characteristics of the bag cells, two clusters of neurosecretory cells whose peptide products initiate and coordinate egg deposition. In particular, the large number of bag cell neurons in a single animal and their relative isolation from other elements of the nervous system facilitate biochemical analyses of the peptides synthesized and released by the cells. Moreover, the episodic nature of bag cell electrical activity, combined with the relative simplicity of the Aplysia nervous system and the stereotypic nature of the behaviors exhibited during egg deposition, provide unusually favorable opportunities for investigating neuropeptide action and function. This review will focus on neuropeptide synthesis and processing in the bag cells and in other organs expressing structurally related genes.
Differentiation of PC12 cells with v‐src: Comparison with nerve growth factor
Authors: Rausch D. M. , Dickens G. , Doll S. , Fujita K. , Koizumi S. , Rudkin B. B. , Tocco M. , Eiden L. E. , Guroff G.
Published: January 2005

The PC12 rat pheochromocytoma cell line is used extensively as a model to study neuronal differentiation. These cells resemble adrenal chromaffin cells, differentiating both morphologically and biochemically when cultured in the presence of dexamethasone, but develop a sympathetic neuron‐like phenotype when cultured in the presence of nerve growth factor. Expression of the protein product of the v‐src oncogene in PC12 cells also induces neurite outgrowth similar to that resulting from nerve growth factor treatment (Alema et al: Nature 316:557–559, 1985). It is thus possible that c‐src or a src‐like tyrosine kinase participates in the signal transduction pathway by which nerve growth factor acts on PC12 cells. In this study a temperature‐sensitive v‐src gene has been introduced into PC12 cells. When cultures of these srctransformed cells are switched from the nonpermissive (40°C) to the permissive (37°C) temperature they elaborate neurites. The differentiation induced by src has been compared with that induced by nerve growth factor by determining whether srctransformed PC12 cells at 37°C exhibit the same biochemical alterations as those induced in PC12 cells treated with nerve growth factor. Neurite extension at 37°C in v‐src—transformed cells, like NGF‐induced differentiation, is accompanied by an increase in the nerve growth factor‐inducible large external (NILE) protein. However, neurite extension in v‐src‐transformed cells is not blocked by the protein kinase inhibitor K‐252a, which completely blocks NGF‐induced neurite extension. Likewise, EGF receptor down‐regulation and the development of saxitoxin and tetanus toxin binding sites are either much reduced or completely absent in src‐differentiated compared with NGF‐differentiated PC12 cells.
v‐src genes stimulate neurite outgrowth in pheochromocytoma (PC12) variants unresponsive to neurotrophic factors
Authors: Eveleth D. D. , Hanecak R. , Fox G. M. , Fan H. , Bradshaw R. A.
Published: January 2005

The NGF‐nonresponsive rat pheochromocytoma PC12 variant nnr5, isolated by Green et al. (J Cell Biol 102:830–843, 1986), responds poorly or not at all to fibroblast growth factor. Transformation of PC‐12nnr5 cells with v–src‐expressing retroviruses results in vigorous neurite outgrowth, similar to that seen in the parent cell line. Thus though the PC12nnr5 cell line has a greatly impaired ability to respond to neurotrophic factors it still may extend neurites. This data is consistent with a model in which PC12nnr5 cells are unable to propagate intracellular second messengers, and this defect may be related to the expression of c‐src gene products.