Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.1.4.37 (CNPase)
 539 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We report the development of a simple and reliable method for the study of demyelination in vitro based on the measurement of 2':3'-cyclic nucleotide 3'-phosphodiesterase in isolated myelin. Using only small quantities of myelin (equivalent to 100 micrograms of myelin protein) the system was tested under conditions that are believed to approximate those found at the site of an inflammatory demyelinating lesion. Treatment with a combination of trypsin, phospholipase A2, and lysophosphatidylcholine was used to evaluate the method. This microsystem has the potential not only for testing the myelinotoxicity of soluble factors but also for investigating the involvement of inflammatory cells in the demyelinating process. Myelin degradation by elicited peritoneal macrophages could be demonstrated at relatively high densities of these cells. Nylon wool purified lymph node T cells from myelin basic protein-primed SJL/J mice, after selective expansion with antigen and interleukin 2, failed to induce any significant myelin breakdown unless a limited number of syngeneic activated macrophages were also present. T cells from mice that had been inoculated with keyhole limpet haemocyanin failed to show any effect. The advantages of this technique over other in vitro systems are that it enables the study of demyelination using syngeneic sources of myelin and defined cell populations.
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PMID:An in vitro micromethod for the quantitative assessment of central demyelination. 245 36

We have analyzed the effects of genetic and epigenetic factors on the steady-state levels of myelin basic protein mRNA and polypeptides during development of mouse oligodendrocytes in culture. Oligodendrocytes were characterized by immunofluorescent staining with antibodies for the following markers: galactocerebroside, myelin basic protein, proteolipid protein, myelin-associated glycoprotein and 2',3'-cyclic nucleotide phosphohydrolase. Oligodendrocytes expressing one or more of these markers first appeared at 3 days in culture and increased to a maximum of 1.5 X 10(5) per brain around 6 days, after which the number remained constant up to 31 days. In medium containing fetal calf serum, accumulation of myelin basic protein polypeptides was delayed relative to in vivo in cultures derived from C57BL/6J, BALB/cJ and DBA/2J inbred mice, but not in cultures derived from C3H/HeJ and AKR/J inbred mice. In medium containing serum from other species or in serum substitute, the temporal expression of myelin basic protein polypeptides in cultures from all the inbred strains was contemporaneous with that in brain. Northern hybridization analysis indicated that the steady-state level of myelin basic protein-specific mRNA in all cultures was regulated similarly to in vivo suggesting that the delayed expression of myelin basic protein polypeptides in some cultures was due to translational and/or post-translational regulation. Analysis of myelin basic protein expression in cultures from informative hybrid and recombinant inbred strains indicated that translational or post-translational expression of myelin basic protein requires trans-acting factors, the inducibility of which is controlled by multiple genetic determinants which segregate independently and are expressed additively.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Expression of myelin basic protein mRNA and polypeptides in mouse oligodendrocytes in culture: differential regulation by genetic and epigenetic factors. 245 46

Patients with Pelizaeus-Merzbacher disease (PM), hemizygous mice with the jimpy mutation (jp/Y), and hemizygous rats with X-linked myelin deficiency (md/Y) share a profound lack of proteolipid protein (PLP) in their central nervous systems (CNS). The peripheral nervous system is normal. These X-linked disorders are associated with or actually caused by the lack of normal oligodendrocytes. Vibratome sections of brain were incubated with antisera to myelin basic protein (MBP), myelin-associated glycoprotein (MAG), 2':3'-cyclic-nucleotide 3'-phosphodiesterase (CNP) (EC 3.1.4.37), PLP, a synthetic PLP-peptide, glial fibrillary acidic protein (GFAP), and transferrin. Reaction product was developed by sequential incubation with biotinylated second antibodies, the avidin-biotin-peroxidase complex (ABC), and diaminobenzidine (DAB) plus hydrogen peroxide as chromogenic substrates. In PM, jp/Y and md/Y, islands of myelin-like structures were revealed by antisera to MBP, MAG, and CNP. Reaction product after application of anti-PLP was absent. Reaction product after anti-PLP-peptide was restricted to infrequent bizarre cells possibly representing abnormal oligodendroglia. The lack of oligodendrocytes in jp/Y and md/Y could also be confirmed by immunocytochemistry for transferrin.
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PMID:Comparative immunocytochemistry of Pelizaeus-Merzbacher disease, the jimpy mouse, and the myelin-deficient rat. 245 99

Using immunofluorescence with a panel of antibodies that recognize antigens expressed by oligodendroglia, the myelin-producing cells of the CNS, at different stages of differentiation from precursor to mature cell, we have investigated the development of cells of this lineage in cryostat sections of rat cerebellum. Our results are consistent with the view that glial precursors, identified by their expression of the ganglioside GD3, arise in the subependymal layers of the 4th ventricle and migrate to their final position in the cerebellum via the superior medullary velum, and to some extent the peduncles. As the cells reach their final destination they make the transition to recognizable galactocerebroside (GC)-expressing oligodendroglia, via a GD3+/GC+ intermediate. The myelin-associated protein 2',3'-cyclic nucleotide 3'-phosphohydrolase (CNP) appears at the same time as GC, whereas myelin basic protein (MBP) is expressed 2-3 days after GC and CNP, immediately prior to myelin formation. A very clear progression of oligodendroglial differentiation was observed from the SMV into the base of the cerebellum, up into the white matter (WM) tracts of the folia, and then away from this central white matter into the granule cell and Purkinje cell layers, and finally the molecular layer. The time delay between the expression of GC, CNP and MBP was the same for oligodendroglia in all of these layers, suggesting the presence of an intrinsic clock controlling the initial expression of these myelin components. The early appearance of CNP in oligodendroglia suggests a role for this protein in the early stages of myelinogenesis.
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PMID:Development of macroglial cells in rat cerebellum. II. An in situ immunohistochemical study of oligodendroglial lineage from precursor to mature myelinating cell. 245 24

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 x 10(5) 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 conjugate 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.
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PMID:Direct microculture enzyme-linked immunosorbent assay for studying neural cells: oligodendrocytes. 245 81

We analyzed the location and abundance of transcripts for the 4 CNS myelin protein genes, myelin basic protein (MBP), proteolipid protein (PLP), myelin-associated glycoprotein (MAG), and 2',3'-cyclic nucleotide phosphohydrolase (CNP), in the mouse cervical spinal cord from the time of rapid myelination until adulthood (8-45 d). In the white matter, maximal levels of transcripts were found for each of the myelin genes at the peak of myelination (8 d). Total MBP and PLP mRNAs stayed high until 20 d and showed a minor decrease thereafter. In contrast, MAG and the MBP exon 2 containing transcripts (coding for the 21.5 and 17 kDa MBP isoforms) decreased sharply between 8 and 20 d, suggesting that high levels of these transcripts are needed primarily during the initiation of myelination. CNP transcripts were less abundant, maintained high expression until 20 d, and then decreased sharply. PLP, MAG, and CNP transcripts were clustered in the oligodendrocyte cell body, while MBP mRNAs were scattered throughout the cell body and processes. In contrast to the white matter, all these myelin specific transcripts in the gray matter showed a marked increase from 8 to 20 d, as did the number of oligodendrocytes identified by CNP immunostaining. MAG transcripts were found in white matter and in satellite and other oligodendrocytes of the gray matter but not in neurons identified by their expression of neurofilament transcripts. The results of our quantitative in situ hybridization study are in good agreement with those of previous molecular studies and provide new information on the cellular and topographic distribution of myelin-specific mRNAs during myelination.
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PMID:In situ hybridization analysis of myelin gene transcripts in developing mouse spinal cord. 246 47

This paper describes the differential expression and localization of myelin components within membrane sheets produced by oligodendrocytes in vitro. In double-labeling experiments using antibodies to the myelin antigens 2',3'-cyclic nucleotide 3'-phosphohydrolase (CNP) and galactocerebroside (GC), the two antigens were coexpressed in at least 95% of oligodendrocytes at all ages examined. A small population of relatively undifferentiated cells expressed one antigen before the other. Within the membrane sheets produced by the cultured cells, CNP and GC are distributed differently. CNP is highly concentrated in cell bodies, in a network of processes extending from the cell body into the sheets, and around the perimeter of the sheets. CNP staining cannot be detected in some areas within the body of the sheet. When present, it is of low intensity. Under our labeling conditions, GC staining is found throughout the membrane sheets, except in the network of veins which are CNP+. GC and myelin basic protein (MBP) staining are seen in similar membrane domains even though GC is a surface component while MBP resides on the cytoplasmic face. Both the timing and localization of CNP immunostaining show that CNP is as early a marker for oligodendrocytes as GC, and support the idea that CNP may play a structural role in the myelin membrane. Double-labeling studies with GC and CNP antibodies also show that the true shape of a cell and the extent of its development are not always revealed by a single antigen. The differential distribution of antigens within membrane sheets illustrates that they contain areas of structural specialization that may reflect the situation in intact myelin.
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PMID:Differential expression of galactocerebroside, myelin basic protein, and 2',3'-cyclic nucleotide 3'-phosphohydrolase during development of oligodendrocytes in vitro. 246 77

Perturbation of myelinogenesis by monoclonal antibodies against galactolipids is being used to study the role of these lipids in oligodendrocyte differentiation. We report here a marked stimulatory effect on oligodendrocyte differentiation when mixed primary cultures initiated from 19-21 day fetal rat telencephala are grown in the presence of a monoclonal antibody against sulfogalactolipids. When such cultures were grown in the presence of the IgM antibody 04 [Sommer and Schachner, Dev Biol 83:311-327 1981], the oligodendrocytes formed aggregates connected by fasciculated processes. Immunofluorescence microscopy and biochemical analyses of treated cultures demonstrated 2-3 fold increases in the fraction of 04-positive cells expressing myelin basic protein, and in the levels of myelin basic protein RNA, myelin basic protein, 2',3'-cyclic nucleotide 3'-phosphohydrolase activity, and 35SO4 incorporation into sulfatide. Greater than 90% of the cells positive for myelin basic protein in treated cultures were in aggregates. The specific activities of oligodendrocyte markers were unaffected in control cultures grown with nonspecific myeloma IgM. Since there was no increase in the total number of 04-positive cells in treated cultures, the increases in the specific activities of the myelin protein markers appears to be due to an increase in the fraction of cells expressing these markers. Time course studies demonstrated that both the rate and extent of oligodendrocyte differentiation were enhanced in treated cultures. These data are discussed with regard to possible mechanisms of the stimulation, considering not only potential direct effects of the antibody on the cell physiology, but also possible indirect effects due to antibody-induced aggregation.
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PMID:Stimulation of oligodendrocyte differentiation in culture by growth in the presence of a monoclonal antibody to sulfated glycolipid. 246 78

Previous work has shown that aggregate cultures prepared from fetal rat telencephalon and grown in a chemically defined medium offer a useful model to study developmental processes such as myelin synthesis. Since compact myelin is formed in these cultures, we investigated the possibility to use this culture system to study demyelinating mechanisms. In particular, we examined the effect of a monoclonal antibody (8-18C5) directed against the myelin/oligodendrocyte glycoprotein (MOG). We found that addition of anti-MOG antibodies and complement to aggregate cultures led to a highly significant decrease in myelin basic protein (MBP) content and 2',3'-cyclic nucleotide 3'-phosphohydrolase (CNP) specific activity. These results indicate that, in our culture system, anti-MOG antibodies have a strong demyelinating effect.
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PMID:Demyelination in brain cell aggregate cultures, induced by a monoclonal antibody against the myelin/oligodendrocyte glycoprotein (MOG). 246 59

Oligodendrocytes, the cells responsible for myelin sheath formation in the central nervous system, were isolated from primary dissociated mixed glial cultures prepared from newborn mouse forebrain, and further cultured in a serum-free defined culture medium. Single and double indirect immunofluorescence using antibodies against the myelin glycolipids, galactocerebroside and sulfatide, and the myelin proteins, myelin basic protein and 2',3'-cyclic nucleotide 3'-phosphohydrolase, was used to investigate the composition of the flat membrane extensions produced by some oligodendrocytes in culture. Galactocerebroside and sulfatide were both expressed on the external surface of the plasma membrane of oligodendrocyte cell bodies and processes and also the membrane expansions. Neither myelin basic protein nor 2',3'-cyclic nucleotide 3'-phosphohydrolase were expressed on the external surface of oligodendrocytes. Myelin basic protein could be localized to the cell body and the membrane expansions but not the major and fine processes. The localization of these myelin components suggests that the expansions have characteristics of the mature myelin membrane. 2',3'-Cyclic nucleotide 3'-phosphohydrolase was found to be localized in the cell body, and in total contrast to myelin basic protein, in the major processes and the fine interconnecting processes, but not the membrane expansions. In some of the cells 2',3'-cyclic nucleotide 3'-phosphohydrolase was present at the outer extremities of the flat membrane sheets, giving the appearance of an extending growth region. Our results thus clearly show that 2',3'-cyclic nucleotide 3'-phosphohydrolase is localized within oligodendrocytes in discrete regions of plasma membranes and suggest that this protein has a possible role in the early stages of myelin formation.
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PMID:Immunohistochemical localization of myelin basic protein and 2',3'-cyclic nucleotide 3'-phosphohydrolase in flattened membrane expansions produced by cultured oligodendrocytes. 247 68


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