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E Yavin

Publications and source records attributed to E Yavin.

At least 91 records · Page 5Linked to original sources
PubMed1985

Polar head group decarboxylation and methylation of phospholipids: an alternate route for phosphatidylcholine formation in cultured neuronal cells.

Decarboxylation of phosphatidylserine (SPG) and methionine-dependent, stepwise methylation of phosphatidylethanolamine (EPG) to form phosphatidylcholine (CPG) were examined in monolayer cultures of rat cerebral cells. Ethanolamine, monomethylaminoethanol, or dimethylaminoethanol nitrogenous bases (N-bases) added to culture medium at millimolar level result each in synthesis of the corresponding phospholipid via a de novo pathway at initial rates of 0.18, 0.30, and 0.36 nmol/h/micrograms DNA, respectively. Addition of methyl-labeled methionine to culture medium at tracer levels or at millimolar concentration enabled measurements of the rates of phospholipid methylation from EPG phosphatidylmonomethylaminoethanol (Me1EPG) and phosphatidyldimethylaminoethanol (Me2EPG) precursors. At tracer doses, the rates of methylation from the above respective phospholipids are 0.45, 1.17, and 1.70 pmol/h/micrograms DNA. At 1 mM methionine, synthesis of CPG proceeds from [14C]EPG or [14C]Me2EPG at initial rates of 8 and 17 pmol/h/micrograms DNA, respectively. Although the latter phospholipid analog can be generated from its monomethyl precursor, methylation of EPG does not result in the accumulation of Me2EPG, suggesting two segregated and metabolically distinct pathways. In the presence of N-bases, of the total [3H]serine incorporated into cellular phospholipids 30-36.5% of labelled SPG is converted into decarboxylation products. The decarboxylation and methylation routes contribute a significant portion of choline from endogenous sources, most likely through conversion of SPG.

Animals↗
PubMed1984

Gangliosides mediate association of tetanus toxin with neural cells in culture.

Somatic neurohybrid SB21B1 cells grown in serum exhibit limited capacity to bind 125I-labeled tetanus toxin and cannot synthesize gangliosides higher than GM2. By 6 h after supplementing the culture medium with pure or mixtures of brain gangliosides, binding of 125I-labeled tetanus toxin to cells increases approximately 8-fold compared to that of nonsupplemented cells. The uptake of added gangliosides is a saturable process and is facilitated by serum removal (2.1-fold) or substitution of growth factors for serum (3.8-fold). Enhancement of tetanus toxin binding to cells depends on the ganglioside species and concentration; GT1b (25 micrograms/ml) is, respectively, two and three times as effective as GD1b and GM1 in increasing toxin binding. Reconstitution of ganglioside-mediated tetanus toxin binding activity is a reversible phenomenon; removal of medium gangliosides causes a 3-fold drop in toxin binding by 24 h, after which an apparent plateau for at least 3 days above the basal level is established. As in cerebral cultures, binding of toxin to ganglioside-supplemented neurohybrid cells exhibits salt and sialidase sensitivity and is enhanced 2.6-fold at 37 degrees C compared to 0-4 degrees C. The resultant temperature-dependent toxin-cell association is sialidase insensitive. Fixation of cells by formaldehyde or treatment of ganglioside-supplemented cells with trypsin has no substantial effect on ganglioside-mediated binding of the toxin. Methanol/chloroform treatment of cells causes a 91.4% loss of binding activity.

Animals↗
PubMed1984

Affinity chromatographic purification and characterization of two iodinated tetanus toxin fractions exhibiting different binding properties.

Highly purified iodinated tetanus toxin preparations separate on ganglioside affinity columns into two distinct (A and B) fractions representing about 20% and 75% of the iodinated toxin, respectively. Fraction A, eluted by 1% NaCl, migrates like native tetanus toxin (150,000 mol. wt) on SDS polyacrylamide gel electrophoresis. It forms an aggregate of molecular weight approximately 360,000 on Sephacryl S-300 gel permeation chromatography in the presence of detergent and contains two isoforms on preparative chromatofocusing. Fraction A binds poorly to neurons in tissue culture or to synaptosomal membrane preparations. It retains, however, its antigenicity and biotoxicity. Fraction B, eluted by 6% NaCl, binds effectively to gangliosides and also to neurons or synaptosome preparations. It has a similar molecular weight and chain composition to the native toxin and displays two isoforms, precipitable during chromatofocusing. Fraction B possesses similar binding, immunological and toxic properties to the original iodinated tetanus toxin. Following excessive iodination (4-6 mCi/mg protein), toxicity can be remarkably reduced. Unlabeled toxin shows a similar chromatographic pattern to the iodinated toxin on affinity columns, suggesting that a large portion (30% by protein and 55% by toxicity) of the toxin has a poor affinity for gangliosides. The molecular pharmacokinetics of tetanus toxin with respect to affinity toward ganglioside-dependent and ganglioside-independent receptors needs re-evaluation.

Animals↗
PubMed1984

Tunicamycin blocks neuritogenesis and glucosamine labeling of gangliosides in developing cerebral neuron cultures.

Fetal cerebral neurons at the initiation of active neurite outgrowth in culture incorporate 4-fold more [3H]glucosamine into glycoproteins than into the cellular lipid fraction. After 8 days or longer, when a well-developed fiber network is apparent, lipids are labeled more extensively than the glycoproteins. Labeling of the latter is inhibited 95% and 89% by 0.5 microgram of tunicamycin per ml added to 1-day-old and 8-day-old cultures, respectively. Labeling of glycolipids is inhibited 30% in 1-day-old and 86% in 8-day-old cultures. Tunicamycin blocks incorporation of glucosamine label into practically all ganglioside species except for a resorcinol-positive, sialidase-sensitive band tentatively identified as GQ1b tetrasialoganglioside (Svennerholm ganglioside nomenclature). It also substantially reduces binding of 125I-labeled tetanus toxin to intact cells. There is 14% and 27% reduction in the total ganglioside sialic acid content in 1-day-old and 8-day-old cells treated for 24 hr with 0.5 microgram of tunicamycin per ml, but no substantial compositional changes are encountered. Tunicamycin blocks neurite outgrowth when added to cells soon after plating but causes no retraction or losses of fibers once the fiber network is established. Therefore, inhibition of neurite outgrowth by tunicamycin is not due to an effect on cellular gangliosides but can be correlated to an inhibition of protein glycosylation.

Animals↗
PubMed1984

Binding of tetanus toxin to somatic neural hybrid cells with varying ganglioside composition.

125I-labelled tetanus toxin interaction with several somatic hybrid cell lines was investigated. Binding of toxin is most effective in NCB-20, followed by NBr-10A, NG108-C15, and SB21-B1 cells. Specific binding of toxin to NCB-20 and SB21-B1 cells is 7- and 60-fold lower, respectively, in comparison to enriched rat cerebral neuron cultures. The NCB-20, NBr-10A, and NG108-C15 clones display a complex ganglioside pattern, including the presence of [N-acetyl-neuraminyl]-galactosyl-N-acetylgalactosaminyl[ N-acetylneuraminyl]-galactosylglucosyl-ceramide (GD1a) and two unidentified [14C]galactose-labelled lipid-soluble compounds, while the SB21-B1 is most abundant in [N-acetyl-neuraminyl]-galactosylglucosyl-ceramide (GM3) and N-acetyl-galactosaminyl-[N-acetyl-neuraminyl]-galactosylglucosyl-c eramide (GM2) gangliosides. None of the cells tested contain measurable levels of [14C]galactose-labelled or resorcinol-positive bands of galactosyl-N-acetyl-galactosaminyl-[ N-acetylneuraminyl-N-acetylneuraminyl]-galactosylglucosyl-ceramide (GD1b) and [N-acetylneuraminyl]-galactosyl-N-acetylgalactosaminyl-[ N-acetylneuraminyl-N-acetylneuraminyl]-galactosylglucosyl-ceramide (GT1b) gangliosides. After 2 h at 37 degrees C a near plateau of toxin association with NCB-20 cells is seen. Binding in low-ionic-strength medium is 1.35-fold higher at 37 degrees C than at 4 degrees C, but is reduced by 21 and 51% at 4 degrees C and 37 degrees C, respectively, in physiologic medium. Treatment of NCB-20 cells with neuraminidase causes a partial loss (29%) of toxin-binding sites. Binding to the hybrid cells is significantly different from that of cerebral cultures with respect to temperature, salt effect, and sensitivity to neuraminidase, suggesting perhaps a different class of receptors for the toxin.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1983

Gangliosides stimulate neurite outgrowth and induce tubulin mRNA accumulation in neural cells.

Morphological changes observed in a somatic neurohybrid clonal cell line SB21B1 are accompanied by a 9 fold increase in the expression of mRNA sequences for tubulin 14 days following the addition of various brain gangliosides to the culture medium. Upon removal of gangliosides, the expression of the tubulin message is restored to near basal levels. Actin mRNA sequences expression is not changed under these experimental conditions. This report documents for the first time, the involvement of gangliosides in the regulation of cellular gene expression for tubulin sequences by a mechanism which may be distinct from that of dibutyryl cyclic-AMP.

Actins↗
PubMed1983

Cell surface and cytoskeletal antigens in cerebral cell cultures after chloroform-methanol delipidation.

Fixation and in situ total delipidation of nerve cells in monolayer cultures have been used for immunofluorescence visualization of cellular antigens. Cells grown on poly-L-lysine-coated coverslips are exposed to a chloroform/methanol (2/1 by vol) solvent mixture for 15 min at -65 degrees C. This step results in the fixation of cells and is accompanied by the complete extraction of cellular lipids. About 3.2% of the total 35S-methionine-labeled trichloroacetic acid-insoluble cellular material is extracted into the organic solvent mixture, demonstrating that aqueous soluble proteins are not significantly extracted under these conditions. A total loss of tetanus toxin binding sites, presumably reflecting the removal of polysialogangliosides from the cell surface, is observed. The accessibility of antibodies against tubulin and against intermediate filaments after this treatment has been investigated. Visualization of these cytoskeletal elements by indirect immunofluorescence and by phase microscopy suggests that the removal of cellular lipids does not affect the apparent cytoarchitecture and that extraction with these organic solvents does not interfere with the staining of tubulin and intermediate filaments, the latter being exclusively present in nonneuronal cells. The advantage of this method of cell fixation and lipid extraction for immunofluorescence of cells in monolayer culture is discussed.

Animals↗
PubMed1983

Temperature-mediated interaction of tetanus toxin with cerebral neuron cultures: characterization of a neuraminidase-insensitive toxin-receptor complex.

Energy-dependent internalization of 125I-labeled tetanus toxin into cultured neural cells is shown to follow an energy-independent binding process. A three-step model, involving receptor-mediated binding followed by sequestration and internalization is proposed. In the first step, binding of toxin is enhanced in appearance under low ionic strength medium, at 0-4 degrees C; it is suppressed, however, with increasing incubation temperature under physiological salt concentrations. Cell-bound toxin is displaced by approximately 35.5% when high-salt medium (physiological concentrations) is added to cells at 0-4 degrees C; the effect is further amplified at 37 degrees C. Addition of disialoganglioside GD1b (1-5 micrograms/ml) also lowers the amount of cell-associated toxin. The fraction of 125I-labeled toxin retained by the cells after exposure to high-salt medium at 0-4 degrees C or after addition of GD1b is operationally defined as sequestered toxin. This second step, characterized by a stable association of the toxin with the neural cells, is affected by both physiological salt and by 37 degrees C conditions. Lastly, an energy-dependent phenomenon of firm association of tetanus toxin with neural cells, compatible with internalization, is described. The toxin residing in this fraction is bioactive and cannot be removed by salts, gangliosides, or by treatment with protease or neuraminidase. Binding, sequestration, and internalization are mutually dependent, as they are all blocked by pretreatment of cells with neuraminidase and by an enhanced energy-independent sequestration event, which results in enhanced tetanus toxin internalization by an energy-dependent process.

Animals↗
PubMed1982

Monoclonal antibodies to the thyrotropin receptor: stimulating and blocking antibodies derived from the lymphocytes of patients with Graves disease.

Human monoclonal antibodies have been generated from heterohybridomas obtained by fusing mouse myeloma cells with peripheral lymphocytes from patients with active Graves disease. This report characterizes four antibodies as presumptive thyrotropin receptor antibodies because they specifically inhibit thyrotropin binding and competitively inhibit thyrotropin-induced cAMP levels in human thyroid cells. Two of these antibodies, 208F7 and 206H3, are representative of autoimmune stimulators in Graves disease sera because they stimulate thyroid function in all assays, including the mouse bioassay; their ability to inhibit thyrotropin-induced cAMP increases in thyroid cells competitively is complemented by more than additive agonism at low (10 pM) thyrotropin concentrations. These stimulating antibodies interact more potently with human thyroid ganglioside preparations than with bovine thyroid or brain gangliosides; in contrast, they are poor inhibitors of 125I-labeled thyrotropin binding to liposomes containing the glycoprotein component of the human thyrotropin receptor. Antibodies 129H8 and 122G3 appear to be representative of inhibiting or "blocking" antibodies in Graves disease sera. Thus they have no intrinsic stimulatory action in assays of thyroid function but rather inhibit thyrotropin activity in the assays tested. These two antibodies do not react with human thyroid gangliosides but are strong inhibitors of thyrotropin binding to liposomes containing the high-affinity glycoprotein component from human, bovine, and rat thyroid membranes. The data unequivocally establish the pluritopic nature of the immunoglobulins in Graves disease and relate individual components or determinants of the thyrotropin receptor structure with specific autoimmune immunoglobulins.

Antibodies, Monoclonal↗
PubMed1982

Gonadotropin-releasing hormone stimulates phospholipid labeling in cultured granulosa cells.

Cultured ovarian granulosa cells from preantral and preovulatory follicles were incubated with [32P]Pi to label endogenous phospholipids. Labeled cells were then incubated with FSH, GnRH, or a GnRH agonist analog [D-Ala6]GnRH (GnRHa), cellular phospholipids were separated by two-dimensional thin layer chromatography, and the radioactivity was determined. Phosphatidylcholine was the major labeled phospholipid accounting for 64% of the total radioactivity. The remaining labeling was distributed among choline plasmalogen (8.4%), phosphatidylinositol (6.3%), lyso phosphatidylcholine (3.7%), phosphatidylethanolamine (3.4%), phosphatidic acid (1.75%), phosphatidylserine (1.65%), and cardiolipin (1.3%). GnRH and its agonist analog GnRHa, but not FSH, increased 32P incorporation into phospholipids by 2-fold. Analysis of the several phospholipids revealed that GnRHa (10(-7) M) increased 32P labeling of phosphatidylcholine and lyso phosphatidylcholine by 1.5- and 2.5-fold respectively, and that of phosphatidic acid and phosphatidylinositol by 5- and 7-fold, respectively, during 60 min of incubation. The natural decapeptide GnRH was 30 times less potent than its agonist analog. Labeling of other phospholipids was not affected by GnRHa treatment, and FSH had no effect on 32P incorporation under similar conditions. The stimulatory effect of GnRHa was blocked by the potent GnRH antagonist [D-pGlu1,pClPhe2, D-Trp3,6]GnRH. The minimal stimulating dose of GnRHa was 10(-12) M, and increased phospholipid labeling could be detected after 10 min of incubation with the analog. These results indicate that phospholipids, in particular phosphatidylinositol and phosphatidic acid, might be involved in the mechanism by which GnRH exerts its gonadal effects.

Animals↗
PubMed1982

Molecular interactions at the cell surface: role of glycoconjugates and membrane lipids in receptor recognition processes.

The present report has described studies using a particular receptor--the TSH receptor--to raise questions concerned with the role of glycolipids and glycoproteins in receptor recognition events and the relevance of lipid modulation of these components in regard to their functional expression. The importance of carbohydrates in other recognition systems will be even more eloquently detailed in subsequent presentations. It is clear, however, that we have gone beyond their recognition as important and are entered into a research stage where questions of how and why are center stage. It is equally clear, as evidenced by the monoclonal antibody data presented herein, that studies of these questions will provide new insight into the mechanism of disease states and will offer new and better diagnostic and therapeutic approaches.

Gangliosides↗
PubMed1981

Tetanus toxin association with developing neuronal cell cultures. Kinetic parameters and evidence for ganglioside-mediated internalization.

Rat cerebral neurons maintained in monolayer culture accumulate 125I-labeled tetanus toxin. Accumulation is receptor-mediated; i.e. it can be prevented by including unlabeled tetanus toxin, gangliosides, or tetanus antitoxin in the incubation medium but not by including tetanus toxoid, high concentrations of serum, or thyrotropin. Accumulation is time-dependent, reaching a plateau after approximately 3 h when 60% of the added toxin is associated with the cells. It is better at 0 degrees C than at ambient temperature and is significantly higher when 0.25 M sucrose replaces physiological salts in a medium containing 5% serum. Unlabeled tetanus toxin, tetanus antitoxin, and tetanus toxoid do not release the accumulated 125I-labeled tetanus toxin to any significant degree; however, gangliosides (50 micrograms/ml) can release 30% of the accumulated 125I-labeled toxin. Treatment of cells with Triton X-100, under conditions where over 90% of the lipids and 70% of the gangliosides are removed, extracts only 15% of the cell-associated 125I-labeled toxin. Evidence is presented that over 50% of the accumulated toxin is internalized in a cellular compartment which is not in immediate equilibrium with the extracellular environment and which is associated with detergent-insoluble cellular constituents. The tetanus toxin accumulated in this compartment has the same gel electrophoretic pattern as the native toxin and is bioactive. The role of gangliosides as potential shuttle vehicles for tetanus toxin internalization is discussed as are the implications of these data to in vitro studies of the pathogenesis of tetanus-induced neurotoxicity.

Animals↗
PubMed1981

Monoclonal antibodies to the thyrotropin receptor: implications for receptor structure and the action of autoantibodies in Graves disease.

Hybridoma cells have been obtained by fusing P3-NS1/1-Ag4-1 mouse myeloma cells with spleen cells from mice immunized with solubilized preparations of the thyrotropin receptor. Five clones were produced that secrete a monoclonal antibody whose binding to thyroid membranes is specifically inhibited by unlabeled thyrotropin. The antibody interacts with functioning thyroid cells in culture but not with nonfunctioning cells; this interaction is prevented by thyrotropin. The antibodies are capable of competitively blocking thyrotropin binding to bovine thyroid membrane preparations; they prevent 125I-labeled thyrotropin binding to a solubilized preparation of the glycoprotein component of the bovine thyrotropin receptor but are unable to inhibit 125I-labeled thyrotropin binding to liposomes containing gangliosides at comparable concentrations. They prevent 125I-labeled thyrotropin binding to rat, bovine, or human (Graves disease) thyroid membrane preparations. They do not stimulate adenylate cyclase activity in thyroid membrane preparations but can inhibit thyrotropin-stimulated iodide uptake by functioning thyroid cells in culture.

Animals↗
PubMed1980

Regulation of phospholipid metabolism in differentiating cells from rat brain cerebral hemispheres in culture: ontogenesis of carrier-specific transport of choline and N-methyl-substituted choline analogs.

Dimethylaminoethanol was studied both as a substrate and as an inhibitor of choline uptake in long-term cultures of foetal rat cerebral hemispheres. A saturable component with an apparent Km of 28 microM and Vmax of 11 pmol/min/microgram DNA for dimethylaminoethanol, was observed. Like choline, dimethylaminoethanol was also taken up by a second, low-affinity component, the apparent Vmax of which was about 102 pmol/min/microgram DNA. Dimethylaminoethanol inhibited the high-affinity but not the low-affinity choline uptake in a competitive manner with an apparent inhibition constant of 6.0 microM. Monomethylaminoethanol (Ki approximately 60 microM) competitively inhibited high-affinity choline transport. At low concentrations hemicholinium-3, but not ethanolamine, effectively inhibited high-affinity uptake of choline and to a lesser degree the uptake of the dimethylaminoethanol. While the high-affinity uptake of both substrates was inhibited by high concentrations of hemicholinium-3 or ethanolamine, the low-affinity system was not affected by hemicholinium-3. From the kinetics of uptake and inhibition patterns of choline and its related analogs, the methyl group seems to play a major role in determining the affinity rate constants for these substrates. The maximum rate of choline uptake via the high-affinity component increases about sixfold during a period of 2 weeks. In the absence of serum the maximum velocity of the high-affinity component is greatly reduced. These observations suggest that the high-affinity choline uptake component is an integral property, and a useful marker, of the developing cerebral cells.

Animals↗