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Bulk separation and long-term culture of oligodendrocytes from adult pig brain. I. Morphological studies 总被引:5,自引:5,他引:0
P. J. Gebicke-Hrter H.-H. Althaus I. Rittner V. Neuhoff 《Journal of neurochemistry》1984,42(2):357-368
Abstract: A method is described by which oligodendrocytes from adult pig brains can be isolated. It results in a cellular preparation suitable for long-term culture. The entire procedure can be accomplished within 2–3 h. The purity of oligodendrocytes ranges between 80 and 95% depending on the Percoll gradient used and on the time in vitro . Yields between 2.5 and 4 × 107 cells per brain and plating efficiencies on the order of 60% make the system very useful for biochemical investigations. It was shown by immunocytochemical studies that oligodendrocytes produce extensive networks of processes, some of them having elaborate membranous expansions. Anti-galactocerebroside (GC) antibodies as well as anti-myelin basic protein (MBP), anti-Wolfgram protein (WP), antiglial fibrillary acidic protein (GFAP), and monoclonal antibodies O1 and O4 are used to identify the cell types and to characterize the cellular composition of the cultures. Anti-GC and O1 are suitable markers for these oligodendrocytes. Both antibodies label similar cells, and the staining intensities are equally strong. In the case of O4, variable staining intensities are observed, and a few additional cells are labeled that are anti-GC− . After 31 /2 weeks in culture, about 60% of the cells can be labeled by anti-MBP. Here too differences in staining intensities are observed. The anti-WP stain is too weak to be defined as positive. The percentage of GFAP+ cells lies in the range 15–20% at maximum. Cells were also mixed into collagen gels. This method appears to be more useful for outgrowth and branching of fibers than are monolayer systems. Drawbacks, however, include limited access for the antibodies and poor recovery of undamaged cells with their fibers. 相似文献
3.
Oligodendroglial proteins labeled with radioactive amino acids were subjected to one- and two-dimensional polyacrylamide electrophoresis. Bands comigrating with myelin proteins, the basic protein (MBP), the proteolipid protein (PLP), and the Wolfgram protein (WP) doublet, were detected by Coomassie Blue staining and by autoradiography. The identity of the MBP and WP in the cellular material is evidenced by immunoblotting with specific antibodies. A comparative study of myelin samples from rat and pig CNS reveals that WP can be detected immunochemically in both species. Different protein patterns, however, are observed. Three protein bands are found with antibodies against the myelin-associated glycoprotein (MAG). The high-molecular-weight component prevails in pig myelin, whereas the medium-molecular-weight component is predominant in rat myelin. Moreover, two protein bands, of molecular weights 35,000 and 33,000 (Ol 1 and Ol 2), are present in high amounts in oligodendroglial particulate material but are not detectable in myelin. These oligodendroglial characteristic proteins are not species-specific, since they are found in preparations of cat oligodendrocytes as well. Activities of cerebroside sulfotransferase (EC 2.8.2.11) are low in freshly isolated cells and increase during the first week of culture. A reverse course of enzyme activities is observed with 2',3'-cyclic nucleotide 3'-phosphohydrolase (EC 3.1.4.37). Values reach a minimum about day 5 in culture and recover their initial values. At day 10 they remain stable until the end of the third week of the culture period. 相似文献
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Nipecotic acid is one of the most potent competitive inhibitors and alternative substrates for the high-affinity -aminobutyric acid transport system in neurons, but the structural basis of this potency is unclear. Because -aminobutyrate is a highly flexible molecule in solution, it would be expected to lose rotational entropy upon binding to the transport system, a change which does not favor binding. Nipecotic acid, in contrast, is a much less flexible molecule, and one would expect the loss of conformational entropy upon binding to be smaller thus favoring the binding of nipecotic acid over -aminobutyric acid. To investigate this possibility, the thermodynamic parameters, G°, H°, and S°, were determined for the binding of -aminobutyrate and nipecotic acid to the high affinity GABA transport system in synaptosomes. In keeping with expectations, the apparent entropy change for nipecotic acid binding (112±13 J·K–1) was more favorable than the apparent entropy change for -aminobutyric acid binding (61.3±6.6 J·K–1). The results suggest that restricted conformation per se is an important contributory factor to the affinity of nipecotic acid for the high-affinity transport system for -aminobutyric acid.This work was conducted when both authors were at the Department of Chemistry, University of Maryland, College Park.Special issue dedicated to Dr. Elling Kvamme. 相似文献
6.
Nadja Zimmer Elisabeth Göttert Joachim Kraus Klaus D. Zang Wolfram Henn 《Human genetics》1993,91(4):317-320
Chromosomal mosaicism in chorionic villus samples (CVS) may arise from different sources, such as clonal diversity within the chorionic tissue or contamination with maternal cells. To determine the origin of karyotyped cells, we compared the immunocytochemical features of mitotic cells in CVS long-term cultures with histological sections of their tissue of origin, i.e. chorionic villi. Immunolabelling of intermediate filaments specific for epithelial cells (cytokeratin) and mesenchymal cells (vimentin) established that mitoses yielded from CVS long-term cultures indeed stem from independently growing clones derived from both the epithelial and mesenchymal parts of the chorionic villi. Thus, mosaicism in CVS cultures may reflect true genetic heterogeneity within the biopsy. However, epithelial chorionic cells undergo in vitro metaplasia leading to co-expression of cytokeratins and vimentin. Fetal-specific immune markers (-HCG and SP1-glycoprotein) are not reliably expressed in CVS cell culture. 相似文献
7.
Savenkoff Claude; da Silva Nadja Lins; Lefevre Dominique; Denis Michel; Rassoulzadegan Fereidoun 《Journal of plankton research》1992,14(6):835-850
Spatial and size distribution of micro-organisms and their ETSactivity has been investigated in Ligurian Sea surface watersalong the Nice-Calvi transect across frontal areas from 18 to37 km offshore (TOMOFRONT 1 and 2 cruises, April 1988 and April-May1989 respectively). Aplastidic and plastidic nanoflagellatesand aplastidic picoflagellates were present in numbers closeto 0.25 x 104 cells ml1, whereas plastidic picoflagellatesaccounted for about half this number. Correlations have beenevidenced between plastidic and aplastidic micro-organisms withinthe same size group, suggesting that they belong to a well-definedecosystem. The highest correlation between total ETS activityand abundance of the considered size groups was observed fornanoflagellates (r = 0.94, n = 22, and r = 0.90, n = 22 foraplastidic and plastidic cells respectively). The importanceof the role of nanoflagellates in surface waters, with respectto the overall ETS activity, was supported by results from sizefractionation which assigned to the 310 µm sizerange a 73.3% contribution to overall ETS activity. Resultsemphasize analysing global ETS activity of natural samples inorder to derive relationships between the different populationspresent in the sampled water. It is suggested that couplingflow cytometry to the ETS approach should be very helpful inthat respect. 相似文献
8.
Poly(ADP-ribose) biosynthesis and suicidal NAD+ depletion following carcinogen exposure of mammalian cells 总被引:1,自引:0,他引:1
R Alvarez-Gonzalez R Eichenberger F R Althaus 《Biochemical and biophysical research communications》1986,138(3):1051-1057
Hepatocytes were found to be remarkably resistant to suicidal NAD+ depletion due to consumption for chromatin-associated poly(ADP-ribose) biosynthesis, which normally follows infliction of DNA damage in mammalian cells. N-methyl-N'-nitro-N-nitrosoguanidine treatment, which depleted NAD+ levels of confluent fibroblasts to about 40% of controls, did not reduce hepatocellular NAD+ pools, although poly(ADP-ribose) concentrations were concomitantly elevated by 21-fold. This differential behavior, demonstrable also with other carcinogens, can be attributed to the different NAD+ biosynthetic capacities of these cells. 相似文献
9.
Escobar Galvis ML Jia J Zhang X Jastrebova N Spillmann D Gottfridsson E van Kuppevelt TH Zcharia E Vlodavsky I Lindahl U Li JP 《Nature chemical biology》2007,3(12):773-778
Heparan sulfate proteoglycans (HSPGs) interact with numerous proteins of importance in animal development and homeostasis. Heparanase, which is expressed in normal tissues and upregulated in angiogenesis, cancer and inflammation, selectively cleaves beta-glucuronidic linkages in HS chains. In a previous study, we transgenically overexpressed heparanase in mice to assess the overall effects of heparanase on HS metabolism. Metabolic labeling confirmed extensive fragmentation of HS in vivo. In the current study we found that in liver showing excessive heparanase overexpression, HSPG turnover is accelerated along with upregulation of HS N- and O-sulfation, thus yielding heparin-like chains without the domain structure typical of HS. Heparanase overexpression in other mouse organs and in human tumors correlated with increased 6-O-sulfation of HS, whereas the domain structure was conserved. The heavily sulfated HS fragments strongly promoted formation of ternary complexes with fibroblast growth factor 1 (FGF1) or FGF2 and FGF receptor 1. Heparanase thus contributes to regulation of HS biosynthesis in a way that may promote growth factor action in tumor angiogenesis and metastasis. 相似文献
10.
Schneider RK Neuss S Stainforth R Laddach N Bovi M Knuechel R Perez-Bouza A 《Differentiation; research in biological diversity》2008,76(2):156-167
Abstract Human mesenchymal stem cells (hMSC) are able to differentiate into mature cells of various mesenchymal tissues. Recent studies have reported that hMSC may even give rise to cells of ectodermal origin. This indication of plasticity makes hMSC a promising donor source for cell-based therapies. This study explores the differentiation potential of hMSC in a tissue-specific microenvironment simulated in vitro . HMSC were cultured air-exposed on dermal equivalents (DEs) consisting of collagen types I and III with dermal fibroblasts and subjected to conditions similar to those used for tissue engineering of skin with keratinocytes. Culture conditions were additionally modified by pre-treating the cells with 5-azacytidine or supplementing the medium with all trans retinoic acid (RA). HMSC were capable of adaptation to epidermis-specific conditions without losing their mesenchymal multipotency. However, despite the viability and evident three-dimensional epidermis-like growth pattern, hMSC showed a persistent expression of mesenchymal but not of epithelial markers, thus indicating a lack of epidermal (trans) differentiation. Further, electron microscopy and immunohistochemical analyses demonstrated that hMSC cultured under epidermis-specific conditions adopted a myofibroblastic phenotype and function, promoted in particular by air exposure. In conclusion, multipotent hMSC failed to differentiate into E-cadherin- or cytokeratin-expressing cells under optimized organotypic culture conditions for keratinocytes but differentiated into myofibroblast-like cells contracting the extracellular matrix, a phenomenon that was enhanced by RA and 5-azacytidine. These results indicate that hMSC might contribute to wound-healing processes by extracellular matrix reorganization and wound contraction but not by differentiation into keratinocytes. 相似文献