Biomedical subjects

S H Yim

Publications and source records attributed to S H Yim.

24 records · Page 2Linked to original sources

PubMed1986

Cultured oligodendrocytes. A role for cell-substratum interaction in phenotypic expression.

Oligodendrocytes can be maintained in two states: nonattached; we call these cells B3.f; morphologically they resemble freshly isolated cells; attached; we refer to the latter as B3.fA. Profound morphological, ultrastructural, and biochemical changes take place upon adhesion to a competent surface (Szuchet, S., Yim, S. H., and Monsma, S. (1983) Proc. Natl. Acad. Sci. U. S. A. 80, 7019-7023). Here we present evidence that the transition from B3.f to B3.fA has important consequences for the expression of myelinogenic properties by these cells. We have examined the incorporation of [3H]leucine, [35S]methionine, and [35S]cysteine into polypeptide chains by B3.f and B3.fA cells from 3 days after isolation up to 8 weeks in culture. Specific antisera against myelin and cytoskeletal proteins were used to identify the newly synthesized proteins. Our results indicate that: overall incorporation expressed as cpm/mg of protein remains essentially constant and independent of the state of adhesion or time in culture; B3.f cells keep a low profile in the synthesis of the major myelin proteins but have a high uptake of precursors into 2',3'-cyclic nucleotide phosphodiesterase, actin, and tubulin; adhesion of oligodendrocytes to a polylysine substratum activates the synthesis and phosphorylation of myelin basic protein, and the synthesis and acylation of proteolipid protein and DM-20; over time in culture there is an increased synthesis and accumulation of these proteins and of myelin-associated glycoprotein. We conclude that B3.f cells exhibit a behavior that is distinct from that of B3.fA cells. Our results are consistent with the notion that upon adhesion to a substratum, oligodendrocytes undergo a transition from myelin-maintaining cells (B3.f) to that of myelin-forming cells (B3.fA). This conclusion is substantiated by the finding of myelin membranes in these cultures.

Animals↗

PubMed1986

Lipid and glycolipid metabolism of cultured astrocytes: a time course study.

Primary cultures of astrocytes free of neurons and containing less than 1% of oligodendrocytes were examined for their ability to incorporate labeled precursors into lipids and glycolipids. At selected developmental stages cultures were double-labeled with either [3H]glycerol and [14C]acetate or with [3H]galactose and Na2[35SO4] for a total of 72 hr. Lipids were extracted with CHCl3/CH3OH, fractionated on a silicic acid column, and further resolved by two-dimensional thin-layer chromatography. It was found that cultured astrocytes actively incorporate acetate and glycerol into various phospholipids; they have very limited ability to utilize galactose and virtually lack the synthetic machinery to use Na2SO4 for the synthesis of sulfated sphingogalactolipids; and their overall lipid metabolism is very distinct from that of oligodendrocytes. It was also found that cultured astrocytes have low levels of 2',3' cyclic phosphodiesterase and glycerol phosphate dehydrogenase activities; the latter is less than one fifth of that in oligodendrocytes.

2',3'-Cyclic-Nucleotide Phosphodiesterases↗

PubMed1986

Mature oligodendrocytes cultured in the absence of neurons recapitulate the ontogenic development of myelin membranes.

We have maintained isolated oligodendrocytes in culture for up to 5 months. Over time cultured cells acquire conspicuous membranous profiles that either emanate from cell processes and perikarya or are apposed to them. These membranous profiles have biochemical and ultrastructural features characteristic of myelin. Because the cells used in this study are post-myelination (cells are isolated from white matter of mature brains), we argue that this is a case of resynthesis of myelin membranes. Because this process takes place in the absence of neurons, we have termed it myelin palingenesis in order to distinguish it from remyelination, a term commonly used to designate reensheathment of axons. Myelin palingenesis may occur seemingly without wrapping and spiraling, but membranes wrapped around oligodendrocyte processes and soma are also observed. Irrespective of the modes of formation, those membranes that are compact have the fine structure of normal myelin. Thus, long-term cultures of mature oligodendrocytes should be a good model system in helping to decipher the molecular and cellular mechanisms involved in myelination and in remyelination.

Animals↗

PubMed1985

A monoclonal antibody that binds to both astrocytes and myelin sheaths.

A monoclonal antibody designated III 5H8 was shown to bind both to astrocytes and to myelin sheaths as studied with immunocytochemical techniques on brain sections and cell cultures. Binding to astrocytes was confirmed by double immunofluorescent labelling of frozen sections and cell cultures with anti-GFAP, and appeared to be sensitive to formalin treatment. Binding to myelin sheaths was confirmed by comparing sections labelled with III 5H8 with sections labelled with antibodies against axons and myelin basic protein as well as by staining of sections of hypomyelinated spinal cord with III 5H8. On immunoblots of separated white matter III 5H8 revealed two bands, while on immunoblots of purified myelin only one band was seen. The findings are discussed with respect to the function of astrocytes in white matter and shared antigenic determinants between astrocytes and oligodendrocytes.

Animals↗

PubMed1984

Characterization of a subset of oligodendrocytes separated on the basis of selective adherence properties.

A subset of oligodendrocytes (B3,f) was isolated by taking advantage of selective cell-substratum interaction. B3,f cells were characterized morphologically, biochemically, and immunocytochemically. Oligodendrocytes were isolated from 4-to-6-month-old lamb brains by a modified version of our published procedure [Szuchet et al, J Neurosci Methods 3:7-19, 1980]. Freshly isolated cells from band III were plated on plastic culture plates at a concentration of 2 X 10(6) cells/ml. Approximately 40% of the cells attached to the plate under these conditions. The remaining cells formed small floating clusters. We refer to the latter as B3,f oligodendrocytes. After 4 to 5 days, the supernatant containing B3,f cells was removed and centrifuged, and the pellet was resuspended in culture medium and replated on polylysine-coated petri dishes. B3,f oligodendrocytes attached to this surface and extended an intricate network of processes. The purity of the cultures, judged by the number of cells staining with a monoclonal antibody against galactocerebroside was 98-99%. This high degree of cell homogeneity was maintained throughout the life of the cultures. B3,f cells appeared to be highly differentiated and remained so in vitro. This is surmised by the expression of oligodendrocytic characteristic functions such as high levels of CNPase activity typically, 5 microM/min/mgP; high incorporation of H2 35SO4 into sulfatides, an overall lipid metabolism that mimics events associated with myelinogenesis [Szuchet et al, PNAS 80:7019-7023, 1983]; the presence, detected immunocytochemically, of myelin-associated glycoprotein and myelin basic proteins. It is concluded that this culture system offers an opportunity for studying the biology of interfascicular oligodendrocytes and their interaction with neurons and/or astrocytes. It also should open up a way of examining the relevance of oligodendrocyte polymorphism.

2',3'-Cyclic-Nucleotide Phosphodiesterases↗

PubMed1983

Lipid metabolism of isolated oligodendrocytes maintained in long-term culture mimics events associated with myelinogenesis.

Oligodendrocytes isolated from ovine white matter according to a published procedure [Szuchet, S., Stefansson, K., Wollmann, R. L., Dawson, G. & Arnason, B. G. W. (1980) Brain Res. 200, 151-164] were cultured for up to 35 days and their capacity to incorporate precursors into lipids was investigated. At various times, cultures were double labeled with [3H]glycerol/[14C]acetate or [3H]galactose/35SO2-4. The cells were harvested 72 hr later and lipids were fractionated using standard procedures. The time course of incorporation for each precursor was distinct. In the days after attachment to substratum, oligodendrocytes preferentially incorporated [3H]glycerol into phospholipids and [14C]acetate into cholesterol while uptake of 35SO2-4 and [3H]galactose into glycolipids was modest. A switch in phospholipid metabolism from preferential incorporation into phosphatidylcholine to incorporation into phosphatidylethanolamine, phosphatidylserine, and phosphatidylinositol occurred at about the 10th day in vitro. After 20 days, uptake of [3H]glycerol into phospholipids and [14C]acetate into cholesterol had stabilized but incorporation of 35SO2-4 into glycolipids had increased. 35SO2-4 incorporation into glycolipids was even greater at 35 than at 20 days. Uptake of [3H]galactose did not change over time. An attempt was made to correlate changes in lipid metabolism with morphologic developments. High incorporation into phospholipids and cholesterol coincided in time with the extensive membrane synthesis required for cell attachment and process extension. Differentiation of these newly formed membranes, as assessed by the incorporation of myelin-characteristic glycolipids, galactocerebrosides, and sulfatides, occurred at a time when an intricate network of processes had already been established. The sequence of metabolic events observed in vitro parallels that observed at the onset of myelinogenesis in vivo. We postulate that mature oligodendrocytes can reenact those early events associated with myelinogenesis.

Acetates↗