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B Zalc

Publications and source records attributed to B Zalc.

At least 73 records · Page 4Linked to original sources

Antisera to the ganglioside GM1 do not have anti-myelin or anti-axon activities in vitro.

Four antisera to the ganglioside GM1 were tested for effects on myelin and axons when applied to mouse spinal cord-dorsal root ganglia explant cultures. None of the antisera to GM1 caused myelination inhibition or demyelination, while an antiserum to galactocerebroside caused both. Antisera to GM1 did not inhibit axonal outgrowth or destroy mature outgrowth zone axons, while an antiserum to a rat brain axolemma-enriched fraction did both. These results suggest that antibodies to GM1 do not have significant anti-myelin or anti-axon activity.

Animals↗

Galactosylceramide: a reliable serum index of demyelination in multiple sclerosis.

Eight patients with multiple sclerosis were followed for several months to determine if serum levels of galactosylceramide, a major lipid component of myelin, correlate with the clinical evolution of the disease. In the patients with the chronic progressive form of multiple sclerosis, galactosylceramide remained undetectable. In the patients with relapsing-remitting multiple sclerosis, there was a good correlation between the elevation of serum galactosylceramide levels and clinical relapses. This serum assay should prove of value in the follow-up of patients with multiple sclerosis.

Adult↗

Developmental expression of myelin proteolipid, basic protein, and 2',3'-cyclic nucleotide 3'-phosphodiesterase transcripts in different rat brain regions.

RNA was extracted from five different rat brain regions during development, starting from embryonic day 15 (E15) until postnatal day 60 (P60). These RNA preparations were analyzed by both Northern and dot blot for their content of 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase), myelin proteolipid protein (PLP), and myelin basic protein (MBP) -specific transcripts. CNPase mRNA was readily detectable at E15 and PLP mRNA at P1 in all brain regions examined. In contrast, expression of MBP mRNA followed a caudorostral gradient. It was first observed at P1 in the mesencephalon and at P9-P11 in the olfactory bulb. Expression of these three transcripts displayed two types of developmental profiles. One was termed biphasic because the specific mRNA level increased regularly and then reached a plateau level. The other developmental profile was termed triphasic, because there was a gradual increase in the level of specific transcripts with a sudden appearance of a sharp peak followed by a decline to a plateau level. When the triphasic pattern was observed, the date of the peak appearance was probe-, but not region-, dependent. It was P15 for CNPase, P18 for MBP, and P21 for PLP. As these peaks occurred at a time during development when myelination was the most active, we postulate the existence of a transient external signal, perhaps neuronal, which would be responsible for this increased amount of myelin-related transcripts.

2',3'-Cyclic-Nucleotide Phosphodiesterases↗

Production and characterization of a mouse monoclonal antibody to the glycolipid asialo-GM1.

The glycosphingolipid asialo-GM1 (aGM1) is a true differentiation antigen of murine lymphoid cells. This glycolipid is highly immunogenic in the rabbit, but the antisera produced shows some cross reactivity with GM1, the naturally occurring sialylated derivative of aGM1. In the present study we examined the ability to raise anti-aGM1 antisera in the mouse. We compared the efficiency of several immunization methods in various strains of mice. The most effective procedure involved repeated intraperitoneal injections of aGM1-cholesterol rich particles in the NZB mouse. Hybrid B cell lines were generated by fusion of mouse myeloma cells with the splenocytes of an NZB mouse immunized with aGM1. The specificity of the antisera produced and of the monoclonal antibody secreted by one of these hybridomas (103HT30) was defined by ELISA and by immunostaining on thin layer chromatograms. The monoclonal antibody 103HT30 is an IgM. It reacted with aGM1 but not with any of the structurally-related ganglioside or neutral glycolipids tested. In particular, 103HT30 monoclonal antibody did not present any detectable cross-reactivity with GM1.

Animals↗

Immunological determination of galactosylceramide level in blood as a serum index of active demyelination.

An enzyme-linked immunosorbent assay (ELISA) to determine the level of galactosylceramide (GalC) in biological fluids is described. The assay uses GalC-coated plastic microtiter plates, with binding of an antibody to GalC detected by a peroxidase-labeled second antibody. The GalC level was directly estimated in the biological samples, without prior extraction, by competition with the coated hapten. This method allows the detection of 62 pmol of GalC (1.2 nmol/ml). Results using this procedure revealed positive sera only among patients suffering a myelin-destructive process: either primary, as in multiple sclerosis, or secondary to brain damage, as during ischemic strokes.

Cerebrosides↗

Regional and developmental estimations of UDP galactose: ceramide galactosyltransferase activity in the rat brain.

The activity of the UDP galactose:ceramide galactosyltransferase (C GalT) was determined during development in the mesencephalon, cerebellum, diencephalon, cerebral hemisphere and olfactory bulb of the rat brain. The level of C GalT activity remained fairly constant at a low level until postnatal day 7 (P7), followed by a simultaneous dramatic increase in all regions (with the exception of the olfactory bulb) tested between P15 and P18, and then by a region-specific rate of decline.

Aging↗

Characterization of anti-cerebroside sulfate antisera using a theoretical model to analyse liposome immune lysis data.

Antisera to the acidic glycolipid cerebroside sulfate (sulfogalactosyl ceramide) were raised in rabbits by several different methods. Reactivity with cerebroside sulfate was detected from complement-mediated lysis of liposomes composed of phosphatidylcholine/cholesterol/cerebroside sulfate and containing the spin label tempocholine chloride as a marker substance. Both cholesterol rich particles and lipid bilayer liposomes containing phosphatidylcholine and cholesterol were effective carriers for cerebroside sulfate, in combination with methylated bovine serum albumin for intravenous immunization, and with Freunds complete adjuvant for subcutaneous immunization. The antisera raised by the different methods were characterized with respect to their cross reactivity with other lipids and the relative concn of specific antibodies and their affinities for cerebroside sulfate using a theoretical model developed earlier [Vistnes A. I. (1984) J. Immun. Meth. 68, 251] for analysis of data from immune lysis of liposomes. Differences in these properties, both of which can affect antibody titer, could be detected for antisera raised by different methods and obtained at different times after immunization. Some of the antisera also reacted non-specifically to varying degrees with other anionic lipids indicating that the anti-cerebroside sulfate antibodies could bind non-specifically to anionic lipids by electrostatic interactions. This suggested that basic amino acid residues may be an important part of the antibody receptor binding site for the glycolipid head group. An important implication of this result is that antibodies raised against anionic glycolipids should be tested for non-specific binding to anionic phospholipids.

Animals↗

[Binding of immunoglobulins of the cerebrospinal fluid of multiple sclerosis patients to oligodendrocytes in culture].

There is often, in multiple sclerosis (MS) an intrathecal synthesis of immunoglobulin G (IgG) with an electrophoretic oligoclonal pattern. The target of those IgG, as well as their possible pathogenic role in the demyelinating process remain unknown and have led to many controversies. We have tested the hypothesis of an oligodendroglial target for those Ig, using a double labelling indirect immunofluorescent assay. Cerebrospinal fluid (CSF) Ig were collected from 28 MS patients (with either progressive or remitting-relapsing disease) and 25 patients with other neurological diseases. Ig binding has been tested on rat and human oligodendrocytes, isolated on a Percoll gradient and maintained 24 to 48 hours in culture. In our experimental conditions, we have never been able to detect any specific binding of MS CSF Ig on those oligodendrocytes, identified by double labelling immunofluorescence with an anti-galactocerebroside antibody.

Animals↗

Schwann cell marker defined by a monoclonal antibody (224-58) with species cross-reactivity. I. Cellular localization.

We have demonstrated by indirect immunofluorescence the cellular localization of a monoclonal antibody (mAb 224-58), produced after immunization of a mouse with human central nervous system (CNS) myelin. Serologically, mAb 224-58 was found to be specific for 3'-sulfomonogalactosylglycolipids, namely 3'-sulfogalactosylceramide (SGC) and 3'-sulfogalactosyl 1-O-alkyl ether 2-O-acylglycerol (seminolipid). This mAb did not bind to SGC-containing tissues such as kidney, liver, spleen, or brain, nor to muscle. However mAb 224-58 did stain positively mouse, rat, and human peripheral nerve sections. In these latter sections, mAb 224-58 was bound to Schwann cell bodies and processes. The specificity of mAb 224-58 for Schwann cells was ascertained on teased rat sciatic nerves and rat Schwann cell cultures. Cells positive for mAb 224-58 were also positive for laminin, and negative for Thy 1-1 antigens both in teased fibers and Schwann cell cultures. In addition, in teased nerve preparations, mAb 224-58-positive cells were also galactosylceramide (GalC)- and SGC-positive. Isolated Schwann cells also expressed 224-58 antigen, even after prolonged time in culture. On testis sections, which contain both SGC and seminolipid, the SGC-positive cells, i.e., the spermatogonia, were always 224-58-negative. But the other germinal cells were 224-58-positive. This suggests that although 224-58 does not discriminate between SGC and seminolipid in serological tests, these lipids in their naturally occurring membrane acquire a spatial configuration that renders them distinguishable to their respective antibody.

Animals↗

Schwann cell marker defined by a monoclonal antibody (224-58) with species cross-reactivity. II. Molecular characterization of the epitope.

A monoclonal antibody (mAb) designated 224-58 (IgM-kappa) has been raised by fusion of Sp2/0-Ag14 mouse hybridoma cell line with spleen cells of a mouse immunized with human brain myelin. This mAb binds specifically to mouse, rat, and human Schwann cells membrane. Serological tests showed that mAb 224-58 reacted with total lipid extract, but not with total protein extract of human myelin. Combination of ELISA, complement fixation, and immunoautoradiographic detection on silica gel TLC allowed us to determine that mAb 224-58 reacted with sulfomonogalactolipids, namely 3'-sulfogalactosylceramide (SGC) and 3'-sulfogalactosyl 1-O-alkyl-ether 2-O-acylglycerol (seminolipid). The fine molecular structure of the epitope recognized by mAb 224-58 was established by studying the cross-reactivity of this mAb toward closely related sulfolipids and by comparing its reactivity after submitting either purified sulfolipids or total lipid extracts to various chemical and enzymatic treatments. The lipidic hapten-binding site to mAb 224-58 is dependent on (1) the sulfoester on carbon 3 of the galactose molecule, (2) the osidic bond, and (3) the carbonyl group of the fatty acid. Interestingly enough, neither the amide bond and the long-chain base nor the OH group of the fatty acid belongs to the antigenic determinant recognized by mAb 224-58.

Animals↗

Oligodendroglial expression and deposition of four major myelin constituents in the myelin sheath during development. An in vivo study.

We have studied the sequence of expression in the oligodendrocyte of four myelin constituents: galactosylceramide (GalC), myelin basic protein (MBP), proteolipid protein (PLP) and Wolfgram protein (W1). These investigations were performed on freshly dissociated cell preparations from mouse olfactory bulb and cerebellum before and during early myelinogenesis. Our data showed that the first myelin antigen to be detected in the oligodendrocyte was GalC, followed 24 h later by W1 and after a 5-day time lag (relative to GalC) by MBP. Expression of PLP occurred shortly after that of MBP. This sequence of events was identical in the cerebellum and in the olfactory bulb, but it commenced earlier in the cerebellum (E18) than in the olfactory bulb (P2). Deposition of these components in the nascent myelin sheaths was studied on tissue sections. These histological preparations showed that the temporal order of deposition of the myelin constituents did not correlate with their order of expression in the oligodendrocyte. As judged both by the intensity of the labeling and the number of positive fibers, W1 and MBP were the first antigens to be deposited, followed by PLP and finally by GalC. Furthermore, this deposition process was initiated immediately after completion of expression of these antigens in the oligodendrocyte.

Animals↗

Multiple sclerosis: rat and human oligodendrocytes are not the target for CSF immunoglobulins.

CSF Ig from 28 patients with MS and 25 patients with other neurologic diseases (OND) were examined for their capacity to bind to rat or human oligodendrocytes in culture. We have used a double-label approach combining CSF Ig with antibodies against galactocerebroside (GalC). As normal human IgG at a concentration higher than 250 micrograms/ml were found to bind nonspecifically to oligodendrocytes in culture, patients' CSFs were used unconcentrated, at a final IgG concentration never exceeding 115 micrograms/ml. In these experimental conditions, we have not been able to detect any fixation of CSF Ig from MS (or OND) patients to rat or human GalC+ oligodendrocytes.

Animals↗

Tamm-Horsfall protein, a kidney marker is expressed on brain sulfogalactosylceramide-positive astroglial structures.

The Tamm-Horsfall (TH) glycoprotein and the acidic glycosphingolipid sulfogalactosylceramide (SGC) have a strictly superimposable localization on kidney tissue sections. The fact that SGC is a prevalent glycolipid in mammalian brain, prompted us to look for the presence of TH in the rat central nervous system (CNS). An antiserum raised against human TH was found to react with rat CNS homogenate in the complement fixation assay. This anti-TH antiserum recognized a rat CNS protein having an identical electrophoretical mobility on SDS polyacrylamide gel electrophoresis (PAGE). Indirect immunofluorescence on rat brain tissue sections allowed us to localize this brain TH cross-reacting material to ependymal cells and astrocytic processes such as the Bergmann fibers or astrocytic feet in contact with either the blood vessels or the meninges. All these astroglial structures are also SGC-positive. Since TH and SGC in the kidney are localized on a membrane that possesses an electrogenic Cl-pump, we propose that the astroglial structures which contain these two molecules are also the site of a Cl-transport system.

Animals↗

Desipramine elicits the expression of opiate receptors and sulfogalactosylceramide synthesis in rat C6 glioma cells.

In the course of our studies on lipidoses induced by amphiphilic drugs, we have investigated the ef- of desipramine, a tricyclic antidepressant, on glial cells in culture. We noted that the addition of desipramine to the culture medium of C6 glioma cells resulted in the modification of the lipid profile of the cell membranes. Of particular interest was the presence, in the desipramine-treated cells, of an additional lipid comigrating on thin layer chromatography with sulfogalactosylceramide (S-GalCer). Addition of radiolabelled sulfuric acid in the culture medium of the desipramine-treated cells resulted in the incorporation of [35S]sulfate in the newly synthesized lipid. Furthermore, this lipid was localized selectively by indirect immunofluorescence using a specific rabbit anti-S-GalCer antibody on the cell surface of desipramine-treated, but not control, C6 cells. Desipramine also increased the activity of 3'-phosphoadenosine-5'-phosphosulfate sulfotransferase (the enzyme responsible for the synthesis of S-GalCer). Since it has been suggested that S-GalCer may be involved in opiate receptors, we looked for opiate binding sites on C6 glioma cells after exposure to desipramine. We found that dihydromorphine was able to bind to the desipramine-treated C6 cell membrane. The binding of [3H]dihydromorphine (180 fmol/mg protein) was stereospecific and had a KD of 30-60 nM. Furthermore, morphine reduced both the basal and isoproterenol-stimulated cyclic AMP levels of the desipramine-treated C6 cells. This effect was blocked by naloxone. In these respects, the opiate binding sites induced after treatment of C6 glioma cells with desipramine fulfill the requirements of a true opiate receptor.

Animals↗

Oligodendrocytes of jimpy mice express galactosylceramide: an immunofluorescence study on brain sections and dissociated brain cell cultures.

Brain sections and dissociated brain cell cultures of jimpy mouse (jp) were investigated for the presence of galactosylceramide (GC) by indirect immunofluorescence. Optic nerve and corpus callosum sections of 26-day-old jp exhibited many GC-positive cells. The GC staining pattern was similar in jp and normal cultures of the same age. These data suggest that the previously observed decreased amount of GC in jp brain is due to the inability of jp oligodendroglia to properly deposit GC in the myelin, while its synthesis is possible.

Animals↗