bluuebunny/crossref_metadata_2025_split huggingface dataset:
Direct microculture enzyme‐linked immunosorbent assay for studying neural cells: Oligodendrocytes
Authors: Gard A. L. , Warrington A. E. , Pfeiffer S. E. 
Published: January 2005

AbstractOligodendrocyte 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

AbstractCathepsin 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

AbstractIn 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

AbstractMuscle 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

AbstractAn 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

AbstractTreatment 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

AbstractCell 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

AbstractPrimary 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

AbstractCyclic 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

AbstractThe 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

AbstractStudies 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

AbstractThe 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

AbstractA 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

AbstractFour 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

AbstractThe 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.