Biomedical subjects

J A Benjamins

Publications and source records attributed to J A Benjamins.

At least 19 recordsLinked to original sources

PubMed2006

Differential effects of Th1, monocyte/macrophage and Th2 cytokine mixtures on early gene expression for immune-related molecules by central nervous system mixed glial cell cultures.

Cytokines secreted within the central nervous system (CNS) are important in the development of multiple sclerosis (MS) lesions. The balance between Th1, monocyte/macrophage (M/M) and Th2 cytokines in the CNS may be pivotal in determining the outcome of lesion development. We examined the effects of mixtures of cytokines on gene expression by CNS glial cells, as mixtures of cytokines are present in MS lesions, which in turn contain mixtures of glial cells. In this initial analysis by gene array, we examined changes at 6 hours to identify early changes in gene expression that represent primary responses to the cytokines. Rat glial cells were incubated with mixtures of Th1, M/M and Th2 cytokines for 6 hours and examined for changes in early gene expression employing microarray gene chip technology. A minimum of 814 genes were differentially regulated by one or more of the cytokine mixtures in comparison to controls, including changes in expression in a large number of genes for immune system-related proteins. Expression of the proteins for these genes likely influences development and inhibition of MS lesions as well as protective and regenerative processes. Analysing gene expression for the effects of various combinations of exogenous cytokines on glial cells in the absence of the confounding effects of inflammatory cells themselves should increase our understanding of cytokine-induced pathways in the CNS.

Animals↗

PubMed2001

Cyclic AMP differentiation of the oligodendroglial cell line N20.1 switches staurosporine-induced cell death from necrosis to apoptosis.

Understanding the regulation of cell death pathways is critical for protecting myelin-producing cells and their associated axons during injury resulting from multiple sclerosis and other degenerative diseases. The immortalized N20.1 oligodendroglial cell line provides a useful model for identifying mechanisms that can be exploited to attenuate cell death in myelin-producing cells and their precursors. In our hands, the N20.1 cell line exhibits different characteristics and morphology depending on temperature (permissive or non-permissive) and the presence of cAMP-elevating agents (Studzinski et al. [1998] Neurochem. Res. 23:435-441; Boullerne et al. [1999] J. Neurochem. 72:1050-1060; Studzinski et al. [1999] J. Neurosci. Res. 57:633-642). Our laboratory previously observed that NO donors cause primarily necrotic death in N20.1 cells grown at permissive temperature, but the NO donor SNP switched a portion of cell death to the apoptic pathway. We have continued our study of apoptotic death in these cells by comparing the effects of staurosporine, a known apoptotic agent, on cells grown at the permissive temperature ("undifferentiated") vs. the non-permissive temperature in the presence of forskolin ("differentiated"). Undifferentiated N20.1 cells exhibit maximal cell death after 24 hr of exposure to 50 nM staurosporine, whereas differentiated cells show delayed cell death, with maximal death seen after 48 hr. Pyknotic nuclei were observed in both growth conditions; however, differentiated cells were protected by caspase inhibitors, whereas undifferentiated cells were not. Increased ssDNA staining and DNA laddering were found following 24-hr staurosporine treatment in the differentiated cells only. These results support the conclusion that N20.1 cells can switch from necrotic to apoptotic cell death when cell division is slowed and cyclic AMP is elevated.

Animals↗

PubMed2001

Role of calcium in nitric oxide-induced cytotoxicity: EGTA protects mouse oligodendrocytes.

Active nitrogen species are overproduced in inflammatory brain lesions in multiple sclerosis (MS) and experimental allergic encephalomyelitis (EAE). NO has been shown to mediate the death of oligodendrocytes (OLs), a primary target of damage in MS. To develop strategies to protect OLs, we examined the mechanisms of cytotoxicity of two NO donors, S-nitroso-N-acetyl-penicillamine (SNAP) and sodium nitroprusside (SNP) on mature mouse OLs. Nitrosonium ion (NO+) rather than NO. mediates damage with both SNAP and SNP, as shown by significant protection with hemoglobin (HbO2), but not with the NO. scavenger PTIO. SNAP and SNP differ in time course and mechanisms of killing OLs. With SNAP, OL death is delayed for at least 6 hr, but with SNP, OL death is continuous over 18 hr with no delay. Relative to NO release, SNP is more toxic than SNAP, due to synergism of NO with cyanide released by SNP. SNAP elicits a Ca2+ influx in over half of the OLs within min. Further, OL death due to NO release from SNAP is Ca2+-dependent, because the Ca2+ chelator EGTA protects OLs from killing by SNAP, and also from killing by the NONOates NOC-9 and NOC-18, which spontaneously release NO. SNP does not elicit a Ca2+ influx, and EGTA is not protective. In comparison to the N20.1 OL cell line (Boullerne et al., [1999] J. Neurochem. 72:1050-1060), mature OLs are (1) more sensitive to SNAP, (2) much more resistant to SNP, (3) sensitive to cyanide, but not iron, and (4) exhibit a Ca2+ influx and EGTA protection in response to NO generated by SNAP.

Animals↗

PubMed2001

Interferon-gamma, tumor necrosis factor-alpha, and transforming growth factor-beta inhibit cyclic AMP-induced Schwann cell differentiation.

Schwann cells differentiate in vivo in response to contact with axons, and cAMP simulates some of these aspects of differentiation in vitro, particularly morphologic changes and expression of certain phenotypic molecules. Unfractionated inflammatory cytokines inhibit cAMP-induced Schwann cell expression of galactolipids (Gal). We sought to identify which cytokines were responsible for this inhibition and to determine whether other phenotypic indicators of Schwann cell differentiation were also affected. Neonatal rat Schwann cells were incubated in vitro with 1 mM 8 Bromo cAMP (8 Br cAMP) with or without the addition of interleukin-1 alpha (IL-1 alpha), IL-1 beta, IL-2, IL-6, tumor necrosis factor-alpha (TNF-alpha), interferon-gamma (IFN-gamma), or transforming growth factor-beta (TGF-beta). Cells were then examined for morphologic changes and for expression of surface Gal and low-affinity nerve growth factor receptor (NGFRp75), employing indirect immunofluorescence. 8 Br cAMP induced Schwann cell upregulation of Gal, downregulation of NGFRp75, and the cells became enlarged and somewhat amorphous and irregular in appearance. Cells treated with IFN-gamma or TNF-alpha alone were more bipolar and more evenly distributed on coverslips than were control cells, whereas TGF-beta alone induced elongated cells often in a swirling pattern. None of the cytokines alone induced upregulation of Gal or downregulation of NGFRp75. TNF-alpha, IFN-gamma, and TGF-beta inhibited the 8 Br cAMP-induced morphologic changes, as well as the upregulation of Gal and downregulation of NGFRp75. The other cytokines had no effects on Gal or NGFRp75 expression. Thus, these three cytokines, which are present in inflammatory lesions in the peripheral nervous system, are capable of inhibiting Schwann cell differentiation.

8-Bromo Cyclic Adenosine Monophosphate↗

PubMed1999

Increased intracellular calcium alters myelin gene expression in the N20.1 oligodendroglial cell line.

Regulation of intracellular Ca(2+) (Ca(i)) plays a central role in cell survival, proliferation, and differentiation. We previously reported that immature oligodendroglia (OLs) are less susceptible than mature OLs to cell death following increases in Ca(i) (Benjamins and Nedelkoska [1995] Neurochem. Res. 21:471-479). The N20.1 murine OL cell line provides a model of an intermediate stage of OL maturation in which to study responses to Ca(i) increases with regard to viability, as well as the expression of mRNAs for myelin basic protein (MBP), proteolipid protein (PLP), DM-20, SCIP, and the immediate early genes ZIF268, c-fos, and c-jun. Cells were treated with the calcium ionophore A23187 or thapsigargin for 1, 3, and 18 hr. A23187 at 1.0 microM had no significant effect on cell detachment or death, whereas thapsigargin at 1.0 microM slightly increased both. With both agents, SCIP, MBP, and PLP mRNA levels were unaffected by 3 hr, but markedly reduced after 18 hours. DM-20 mRNA levels remained unchanged at both time points. With both agents, ZIF268, c-fos, and c-jun mRNA levels were unaffected after 1 hr; c-jun mRNA levels showed a significant increase after 3 hr of thapsigargin treatment. Thus, in N20.1 cells, increased calcium affects the IEG c-jun first, SCIP is coordinately decreased with MBP and PLP mRNAs at a later time point, and DM-20 message is under different regulation than PLP. J. Neurosci. Res. 57:633-642.

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PubMed1999

Synergism of nitric oxide and iron in killing the transformed murine oligodendrocyte cell line N20.1.

Nitric oxide (NO) produced in inflammatory lesions may play a major role in the destruction of oligodendrocytes in multiple sclerosis and experimental allergic encephalomyelitis. The transformed murine oligodendroglial line N20.1 is much more resistant than primary oligodendrocytes to killing by the NO generator S-nitroso-N-acetyl-DL-penicillamine (SNAP). This observation prompted investigation of the mechanisms leading to cell death in the N20.1 cells and comparison of SNAP with another NO donor, sodium nitroprusside (SNP). We observed that N20.1 cells were 30 times more sensitive to SNP than to SNAP. The specific NO scavenger 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (PTIO) protected against SNP only, not against SNAP. However, dithiothreitol protected against both SNAP and SNP, indicating that S-nitrosylation of cysteines plays a major role in the cytotoxicity of both NO donors. We did not observe any formation of peroxynitrite or increase of Ca2+ concentration with either SNAP or SNP, thus excluding their involvement in the mechanisms leading to N20.1 cell death. Based on two observations, (a) potentiation of the cytotoxic effect of SNP when coincubated with ferricyanide or ferrocyanide, but not sodium cyanide, and (b) protection by deferoxamine, an iron cyanide chelator, we conclude that the greater sensitivity of N20.1 cells to SNP compared with SNAP is due to synergism between NO released and the iron cyanide portion of SNP, with the cyanide accounting for very little of the cytotoxicity. Finally, SNP but not SNAP induces some apoptosis, as shown by DNA laddering and protection by a caspase-3 inhibitor. These results suggest that low levels of NO in combination with increased iron content lead to apoptotic cell death rather than the necrotic cell death seen with higher levels of NO generated by SNAP.

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PubMed1998

TNF-alpha and TGF-beta act synergistically to kill Schwann cells.

Interactions between cytokines and Schwann cells (SC) are important in development, repair, and disorders of the peripheral nervous system (PNS). Tumor necrosis factor-alpha (TNF-alpha) and transforming growth factor-beta (TGF-beta) are two prominent cytokines which may be involved in these processes and their gene products are upregulated in some experimental neuropathies. This study focuses on the in vitro effects of these cytokines, both singly and in combination, on cultured SC. Expression of both Type I and Type II TNF-alpha receptors was demonstrated on the SC surface by immunocytochemistry. Treatment of SC with a combination of TNF-alpha plus TGF-beta causes significant detachment and cell death while treatment with each cytokine alone is not significantly cytotoxic. When compared with control cultures, SC treated with the combination of cytokines exhibit an increase in the number of cells with condensed nuclei and evidence of DNA fragmentation, characteristics consistent with cells undergoing programmed cell death. Thus, TNF-alpha plus TGF-beta induce SC loss of adhesion which is predominantly due to cell death. Apoptotic mechanisms are likely to contribute to some extent to this cell death. These findings provide in vitro evidence to support the hypothesis that cytokines can directly damage SC in PNS disorders.

Animals↗

PubMed1998

Binding of cholera toxin B subunit: a surface marker for murine microglia but not oligodendrocytes or astrocytes.

GM1 ganglioside is a receptor for the B subunit of cholera toxin. In lymphocytes, B subunit elicits an influx of extracellular Ca++ (Dixon et al., 1987). To investigate this signaling pathway in glia, we assessed the presence of GM1 ganglioside on the surface of cultured murine central nervous system (CNS) glia by binding of fluorescein-labeled B subunit. B subunit binding was compared to binding of peanut agglutinin, wheat germ agglutinin, and Bandeiraea (Griffonia) simplicifolia lectin (BSL)I, a microglial marker. Antibodies to glial fibrillary acidic protein, A007/O4 antigens, and galactocerebroside were used to identify astrocytes, immature oligodendrocytes (OLs) and mature OLs, respectively. Binding patterns differed based on cell type and developmental stage. Wheat germ and peanut agglutinins bound to the surface of microglia, astrocytes, and immature OLs; neither lectin bound to any significant extent to the surface of membrane sheets of mature OLs, although wheat germ agglutinin was rapidly endocytosed. Cells identified as microglia by BSL I binding and morphology were the only cells to stain brightly on the surface with B subunit. Thus, surface GM1 ganglioside appears to be a highly enriched marker for microglia in these mixed glial cultures. The effects of B subunit on intracellular Ca++ were examined by laser cytometry in glial cultures loaded with Indo-1. No Ca++ responses were observed in microglia. Mature OLs were examined for Ca++ responses to B subunit before and after surface levels of GM1 ganglioside were increased by incubation with exogenous GM1 ganglioside. Again, no Ca++ responses were observed. Thus, cultured microglia and mature OLs do not have the GM1-mediated signal transduction pathway seen in lymphocytes. However, the presence of GM1 ganglioside on microglia may play a role in giving rise to antibodies to this glycolipid in some CNS inflammatory diseases.

Animals↗

PubMed1998

Effects of cyclic AMP on expression of myelin genes in the N20.1 oligodendroglial cell line.

The N20.1 immortalized cell line has several characteristics of differentiating oligodendrocytes (OLs), including expression of the glycolipids galactocerebroside (GalC) and sulfatide, and the myelin proteins CNPase and myelin basic protein (MBP) (1,2). Addition of 1-100 microM forskolin to elevate cyclic AMP (cAMP) levels changed cell morphology from irregular and flattened to a more rounded birefringent cell with multiple branched processes. GalC and sulfatide were detected immunocytochemically after permeabilization in the untreated cells and levels appeared to increase slightly following exposure to forskolin. Further analysis showed that most of the glycolipid was internal, with virtually no detectable levels on the cell surface in untreated cells and a very slight change following treatment with forskolin. Synthesis of the two lipids as measured by [H3]galactose incorporation doubled within 24 hours of treatment with forskolin. Levels of message for UDP-galactose: ceramide galactosyl transferase (CGT), a key enzyme in the synthesis of GalC and sulfatide, were compared with those of MBP and proteolipid protein (PLP), before and after elevation of cAMP. No changes were observed in levels of mRNA for CGT and PLP after 24 hours, with a possible increase by 48 hours. In contrast, levels of MBP message dropped precipitously by 24 hours; this was accompanied by an increase in levels of message for suppressed cAMP-inducible POU (SCIP). Thus CGT transcription is regulated independently of MBP and SCIP in N20.1 cells. Analysis of MBP levels by immunocytochemistry and Western blot showed little or no change in protein levels at 24 and 48 hours, in contrast to the sharp decrease in message levels by 24 hours, indicating a relatively long half life for MBP in this cell line. Thus, the N20.1 cells are an informative model for examining regulation of expression of myelinotypic proteins and GalC, as well as the transport of this lipid to the plasma membrane.

Animals↗

PubMed1998

Antibodies to glycolipids and cholera toxin B subunit do not initiate Ca++ signaling in rat Schwann cells.

Antibodies to glycolipids have been implicated in the pathogenesis of several immune-mediated PNS demyelinating diseases. This study focuses on antibodies to galactocerebroside (GalC) and sulfatide and on the B subunit of cholera toxin (CTB), which reacts with GM1 ganglioside, to examine whether these agents have any direct effects on Schwann cells (SC) as measured by Ca++ responses. While surface levels of GalC and sulfatide were markedly upregulated by 8 Br-cAMP treatment, as reported by others, very little expression of surface GM1 ganglioside was detected with or without 8 Br-cAMP treatment. Schwann cells, under either condition, showed no changes in intracellular Ca++ levels when exposed to purified monoclonal antibodies reacting with GalC or sulfatide. Thus upregulation of surface levels of GalC or sulfatide does not lead to antibody-induced Ca++ influx, in contrast to previous findings in mature oligodendrocytes (OLs) exposed to antibodies to GalC. Further, cross-linking with one of the antibodies (R-mAb) did not produce Ca++ responses. No Ca++ responses were elicited by CTB in Schwann cells either with or without 8 Br-cAMP treatment. Since surface binding of CTB was very low and sparsely punctate in Schwann cells +/- 8 Br-cAMP, we tested whether increasing levels of GM1 ganglioside on the surface would lead to induction of a Ca++ signaling pathway, as reported for fibroblasts. GM1 ganglioside on the surface of SC was markedly increased by exposing cells to exogenous GM1 ganglioside, but no Ca++ responses were observed in the treated cells. Thus undifferentiated or partially differentiated SC lack the glycoconjugate-mediated Ca++ signaling pathways found in mature OLs or fibroblasts.

Animals↗

PubMed1996

Epigenetic factors up-regulate expression of myelin proteins in the dysmyelinating jimpy mutant mouse.

Proteolipid protein (PLP) is a major structural component of central nervous system (CNS) myelin. Evidence exists that PLP or the related splice variant DM-20 protein may also play a role in early development of oligodendrocytes (OLs), the cells that form CNS myelin. There are several naturally occurring mutations of the PLP gene that have been used to study the roles of PLP both in myelination and in OL differentiation. The PLP mutation in the jimpy (jp) mouse has been extensively characterized. These mutants produce no detectable PLP and exhibit an almost total lack of CNS myelin. Additionally, most OLs in affected animals die prematurely, before producing myelin sheaths. We have studied cultures of jp CNS in order to understand whether OL survival and myelin formation require production of normal PLP. When grown in primary cultures, jp OLs mimic the relatively undifferentiated phenotype of jp OLs in vivo. They produce little myelin basic protein (MBP), never immunostain for PLP, and rarely elaborate myelin-like membranes. We report here that jp OLs grown in medium conditioned by normal astrocytes synthesize MBP and incorporate it into membrane expansions. Some jp OLs grown in this way stain with PLP antibodies, including an antibody to a peptide sequence specific for the mutant jp PLP. This study shows that: (1) an absence of PLP does not necessarily lead to dysmyelination or OL death; (2) OLs are capable of translating at least a portion of the predicted jp PLP; (3) the abnormal PLP made in the cultured jp cells is not toxic to OLs. These results also highlight the importance of environmental factors in controlling OL phenotype.

Animals↗

PubMed1996

Release of intracellular calcium stores leads to retraction of membrane sheets and cell death in mature mouse oligodendrocytes.

The ability of mature oligodendrocytes (OLs) to recover from insult is important in repair of damage following demyelination. Since regulation of Ca2+ levels within cells plays a critical role in function and survival, this study investigates the effects of changes in cytoplasmic Ca2+ on the viability of cultured mouse OLs and their ability to maintain membrane sheets. Mature OLs in culture respond rapidly to the calcium ionophore A23187 and promptly return to resting Ca2+ levels when the ionophore is removed. Longer exposure to 0.1-1.0 microM A23187 leads to microtubule disruption, membrane sheet retraction and eventual cell death; nuclear lysis occurs in many of the OLs, as reported by Scolding, et al. (1) for rat OLs. In our cultures, mature OLs were more susceptible to nuclear lysis than were immature OLs or astroglia. Release of intracellular Ca2+ stores with thapsigargin at 5-10 microM also leads to retraction of membrane sheets. Following 6 hours of continuous exposure to thapsigargin, the effects on membrane sheets are reversed over the next 12 hours. After 18 hours of continuous exposure to thapsigargin, only occasional nuclear lysis is observed, but a number of the mature OLs show signs of DNA fragmentation, indicating that apoptotic death is occurring. Our results suggest that mature OLs cannot survive a prolonged influx of extracellular calcium as readily as immature OLs and astroglia, but have mechanism to withstand similar increases in cytoplasmic Ca2+ following sustained release of intracellular stores.

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PubMed1994

Analysis of myelin proteolipid protein and F0 ATPase subunit 9 in normal and jimpy CNS.

Membrane fractions and chloroform-methanol (C-M) extracts of jimpy (jp) and normal CNS at 17-20 days were examined by immunoblot and sequence analysis to determine whether myelin proteolipid protein (FLP) or DM-20 could be detected in jp CNS. No reactivity was detected in jp samples with several PLP antibodies (Abs) except with one Ab to amino acids 109-128 of normal PLP. Proteins in the immunoreactive bands approximately 26 M(r) comigrating with PLP were sequenced for the first 10-12 residues. A sequence corresponding to PLP was found in normal CNS, as expected, but not in the band from jp CNS. Our results provide no evidence for an aberrant form of PLP in jp CNS at 17-20 days. This and other studies suggest that the abnormalities in jp brain are not due to toxicity of the mutant jp PLP/DM-20 proteins. Interestingly, a sequence identical to the amino terminus of the mature proton channel subunit 9 of mitochondrial F0 ATPase was detected in the immunoreactive bands approximately 26 M(r) in both normal and jp samples. This identification was supported by reactivity with an Ab to the F0 subunit and by labeling with dicyclohexylcarbodiimide (DCCD). In contrast to PLP isolated from whole CNS, PLP isolated from myelin was devoid of F0 subunit 9 based on sequence analysis and lack of reactivity with an Ab to the F0 subunit, yet still reacted with DCCD. This finding rules out the possibility that contaminating F0 ATPase gives rise to the DCCD binding exhibited by PLP and confirms the possibility that PLP has proton channel activity, as suggested by Lin and Lees (1,2).

Absorption↗

PubMed1994

Maintenance of membrane sheets by cultured oligodendrocytes requires continuous microtubule turnover and Golgi transport.

Oligodendrocytes in murine shakeoff cultures elaborate extensive membrane sheets containing networks of microtubules. Several membrane components, including proteolipid protein (PLP) and sulfatide, are transported through the Golgi en route to the plasma membrane or myelin (1,2). This transport is essential for membrane assembly, but its role in continuing maintenance of the sheets is not known. We examined the stability of the membrane sheets following microtubule stabilization with taxol or block of transport into the Golgi with brefeldin A. Within one to three hours, both agents had marked effects on the membrane sheets. While some oligodendrocytes maintained regions of normal membrane sheets, many showed retraction of the sheets, with the majority now exhibiting multiple processes rather than sheets. The distribution of sulfatide, PLP and tubulin in cell bodies, processes and sheets was altered in treated cells, as analyzed by immunocytochemical staining with antibodies to these components. The Golgi apparatus also showed reorganization in the presence of taxol, as visualized by binding of wheat germ agglutinin, a lectin with high affinity for distal Golgi vesicles. All of these effects were reversible when the agents were removed after 3 hours. Thus, maintenance of membrane sheets by oligodendrocytes in culture is a dynamic process, requiring ongoing microtubule turnover and transport of molecules through the Golgi.

Animals↗

PubMed1992

Entry of newly synthesized gangliosides into myelin.

Ganglioside synthesis and transport to myelin was studied in brainstem slices prepared from 19-21-day-old rats. The slices were incubated for up to 2 h in the presence of [3H]glucosamine to label primarily the hexosamine portion of complex gangliosides. The amount of radioactivity incorporated into gangliosides during slice incubations was only 10-15% of the amount of the label incorporated during in vivo labeling of brainstem gangliosides using equivalent amounts of [3H]glucosamine. Among individual gangliosides this inhibition was greater for the more complex gangliosides. When labeled gangliosides were isolated from homogenate and myelin fractions prepared from brain slices, the complex total gangliosides of both fractions showed a lag in labeling kinetics but with a lower specific radioactivity for the myelin fraction, reflecting the larger pool size and slower turnover rate exhibited by myelin components. Chase experiments showed that more complex gangliosides in homogenate exhibited almost no effect of chase after 30 min. Addition of the Golgi-disrupting agent monensin to slice incubations inhibited the labeling of all gangliosides except GM3, GM2, and GD3, and transport to myelin of all complex gangliosides except GM2. These results show that a monensin-sensitive mode of transport is responsible for the translocation of most newly synthesized gangliosides into myelin.

Animals↗

PubMed1992

Caprine beta-mannosidosis: regional differences in deficits of CNS myelin proteins.

Caprine beta-mannosidosis is an autosomal recessive disorder characterized by marked deficiency of beta-mannosidase activity, accumulation of oligosaccharides, and pathologic changes involving prominent dysmyelination. The myelin deficits show marked regional variation, with spinal cord mildly affected and many brain regions severely affected by morphologic criteria. In this study, levels of myelin basic protein (MBP) and proteolipid protein (PLP) were measured by immunoblotting in samples prepared from spinal cord, brainstem and cerebral hemispheres of normal and affected goats at 2-4 days (newborns) and 2-4 weeks of age. In affected goats, total levels of MBP in spinal cord were normal, while PLP levels were 60-70% of normal at both ages. In contrast, PLP and MBP in brainstem and cerebral hemispheres were severely decreased at both ages, with levels of PLP 10-13% and MBP 25-29% of normal in newborns, and generally more reduced at 2-4 weeks. When myelin fractions were isolated on 0.32/0.85 M sucrose gradients, yields were about 38 and 25% of normal in spinal cord at the two ages, but less then 3% of normal in brainstem. Yields of myelin-like fraction were decreased as well, but to lesser extents than yields of myelin. Myelin from spinal cord had a normal composition with regard to PLP and MBP content, while the myelin fraction from brainstem was markedly deficient in both proteins. This suggests formation of myelin with a very abnormal composition in brainstem, or inclusion of large amounts of membranes other than myelin in this fraction. The more severe deficits in brainstem and cerebral hemispheres compared to spinal cord are consistent with morphologic observations.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1991

Galactocerebroside and sulfatide independently mediate Ca2+ responses in oligodendrocytes.

Galactocerebroside (GalC) and sulfated galactocerebroside (sulfatide) are sphingolipids highly enriched in myelin. The binding of antibodies reactive with either sulfatide or GalC to cultured oligodendrocytes causes a Ca2+ influx, followed by microtubule depolymerization; however, antisulfatide is less effective than anti-GalC in altering cytoskeleton. Typical Ca2+ responses are delayed for both antibodies but are transient for sulfatide-reactive antibodies in contrast to the sustained responses previously reported for anti-GalC (Dyer and Benjamins, J Cell Biol 111: 625-633, 1990). Approximately one-half as many oligodendrocytes respond to sulfatide-reactive antibodies (about 39%) as to anti-GalC (about 75%). Subpopulations of oligodendrocytes were identified that responded to neither antibody, only one antibody, or both antibodies, indicating that sulfatide and GalC independently mediate Ca2+ responses. These results suggest that sulfatide and GalC have different physiologic roles in regulating elaboration of myelin membrane by oligodendrocytes in vivo and support the possibility that viral or immune attack via GalC or sulfatide on oligodendrocytes may mimic normal signals in a manner that disrupts the sequence of events that coordinates myelination or maintenance of myelin in vivo.

Animals↗

PubMed1990

Phagocytosis of peripheral nerve myelin in vitro: effect of antibody.

We have previously shown that antisera to whole CNS myelin, whole PNS myelin, galactocerebroside (GC), and myelin basic protein (MBP) promote the uptake of CNS myelin by cultured macrophages, and stimulate the conversion of myelin lipids to cholesterol ester and triglycerides. Here we report the results of similar studies using PNS myelin purified from the rat sciatic nerve. Antisera to whole CNS myelin, whole PNS myelin, GC, and MBP preincubated with 14C-labeled PNS myelin increased the production of radioactive cholesterol ester by macrophages in culture to a level about twice that with preimmune serum, and five to six times that of untreated myelin. The amounts of [14C]triglyceride were similarly increased with these antisera, whole P0 and P2 antisera had little or no effect. IgG prepared from the antisera stimulated lipid metabolism to almost the same extent, while heating the antisera did not decrease the stimulatory effect, indicating that myelin was opsonized by IgG, but not likely by complement. With a few exceptions, the four active sera and their IgGs promoted the macrophage metabolism of CNS and PNS myelin almost equally. The cultured macrophages converted about 3% of untreated CNS myelin and about 6% PNS myelin cholesterol to cholesterol ester. Under phase contrast microscopy it was noted that vesicles of CNS myelin appeared to bind individually to macrophages, whereas PNS myelin vesicles tended to self-associate to form large clumps which were found to macrophages. Binding studies showed PNS myelin to be bound more firmly to macrophages than CNS myelin.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗