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E Yavin

Publications and source records attributed to E Yavin.

At least 55 records · Page 3Linked to original sources
PubMed1993

Elongation, desaturation, and esterification of essential fatty acids by fetal rat brain in vivo.

Tracer amounts of either [1-14C]linolenic (18:3n-3, LNA), or [1-14C]linoleic (18:2n-6, LA) acids were intracranially injected into 19- to 20-day-old rat fetuses, and the time course of the in vivo formation and esterification of their long chain polyenoic metabolites was determined for up to 20 h. A rapid disappearance of free LNA and LA, with apparent half-lives of 60 and 40 min, respectively, was noticed. One hour after LNA injection, 32.3% and 14.3% of the total brain radioactivity was found in the neutral glyceride (NG) and phospholipid (PL) fractions, respectively. After 20 h, PL radioactivity attained a level of 75%. Phosphatidylcholine (PC), diacylglycerol (DG), and triacylglycerol (TG) constituted 40%, 23%, and 9% of the total brain label at 1 h, and 35%, 10% and 14% at 20 h, respectively. Ethanolamine-containing PL (including plasmalogen) radioactivity accounted for about 10% up to 6 h and increased nearly 3-fold at 20 h, primarily due to an increase in the amount of labeled docosapentaenoic and docosahexaenoic acids (DHA), the elongation-desaturation products of LNA. A similar pattern of incorporation into NG and PL fraction was observed after the administration of [1-14C]LA. After 1 h, PC, DG, and TG species constituted 23%, 10%, and 23% of the total brain radioactivity, whereas after 20 h it accounted for 44%, 6%, and 10%, respectively. Although radioactivity in the ethanolamine PL also increased substantially from 4% at 1 h to 29% at 20 h, the main labeled fatty acid in this fraction was LA. Labeled arachidonic acid (AA) constituted 42.7% of the total radioactivity in phosphatidylinositol (PI) at 20 h. At this time, it comprised 12.5% and 14% of the total radioactivity in PC and ethanolamine PL, respectively, suggesting a high degree of esterification selectivity. Comparison of the total amounts of LA and LNA and their corresponding labeled AA and DHA metabolites in brain and liver after 3 and 6 h indicated that the contribution of liver metabolism to the elongation-desaturation under these conditions was negligible. One hour after intracerebral injection of [3H]DHA (22:6n-3) or [3H]AA (20:4n-6), 29.2% and 12% of total radioactivity, respectively, was found in the ethanolamine PL while 20% and 40% was incorporated in PC, respectively. PI labeling by [3H]AA was 6- to 8-fold higher than that seen in the presence of DHA. A high percent of radioactivity (26.9% and 18.2%) was found in DG and TG species.(ABSTRACT TRUNCATED AT 400 WORDS)

Acetyltransferases↗
PubMed1992

Intracellular calcium levels regulate the actions of nerve growth factor on calcium uptake in PC12 cells.

The uptake of divalent cations and the intracellular concentration of calcium in PC12 cells were studied by flow cytometric analysis using the calcium-sensitive dye, Fluo-3, under a variety of conditions. In particular the actions of nerve growth factor were analyzed. The data show that nerve growth factor stimulates the uptake of divalent cations and increases the intracellular calcium levels of cells attached to collagen-coated plates. The data further indicate that nerve growth factor-dependent increases in the uptake of divalent cations become less pronounced as the intracellular concentration of calcium increases. Intracellular calcium levels increase upon detachment of the cells from the plates and also with increasing cell density. Studies on the uptake of 45calcium confirmed the influence of intracellular calcium levels on nerve growth factor-stimulated calcium uptake. Thus, the effect of nerve growth factor on the uptake of divalent cations is dependent on the calcium levels in the cells, perhaps explaining why previous studies in this field have provided inconsistent results.

Animals↗
PubMed1992

Regulation of arachidonic acid metabolism in the perinatal brain during development and under ischemic stress.

Oxygen deprivation following cessation of blood flow to vital organs such as brain, heart, and kidney is a ubiquitous human disease, invariably leading to devastating consequences. Studies in experimental models support the contention that membrane permeability is altered, ion fluxes impaired, and energy stores depleted under these circumstances. Certain lipids such as diglycerides (DG) and arachidonic acid (AA), both of which are important cellular second messengers, appear to increase during ischemia. At this point, the contribution of these and other lipids to cell deregulation, loss of function, and ultimate death has not been clarified because no precise link between lipid alterations as detected in ischemia and subsequent cellular processes has been made. In this report we examine the origin of lipid-derived second messengers in fetal rat brain prelabeled with [3H]AA and study the fate of various lipids upon obstruction of the fetal-maternal circulation. The data support the possibility of a phospholipase A2-mediated deacylation of poly-phosphoinositides (poly-PI) to form free AA and a phospholipase C-mediated hydrolysis of PC to form DG during ischemia.

Animals↗
PubMed1992

Isolation and characterization by cell density adjustment of a PC12 pheochromocytoma variant with altered Ca2+ homeostasis.

Rat PC12 pheochromocytoma cells loaded with the fluorescent Ca2+ dye fluo-3 or indo-1 and scanned fluorimetrically on a cell sorter apparatus showed a rapid cell density-dependent increase in free cytosolic calcium concentration [Ca2+]i when maintained in suspension cultures. Cell adhesion, measured under a defined set of conditions, was low when cells were seeded at 1.5 x 10(4) cells/ml but reached maximal levels after addition of A23187 calcium ionophore. A six to sevenfold increase in cell density mimicked the effect of the ionophore. Densities above 2 x 10(6) cells/ml caused a decrease in cell adhesion, which was further reduced by the addition of A23187. BAPTA, AM (1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid) and nifedipine (10 microM each), partially inhibited cell attachment (34% and 44% reduction), but at 0.25 microM and 1 microM, respectively, they enhanced attachment (46% and 67% increase). The data suggest that a certain permissive level of [Ca2+]i, attained by either increasing cell density or by the presence of a calcium ionophore, is sufficient for maximal cell adhesion. Above the permissive level, manipulation of [Ca2+]i either by altering cell density or by the addition of calcium blocking agents in high concentrations results in a significant reduction in cell adhesion. Based on these observations, we were able to isolate a biochemically and morphologically distinct cell population. The variant, designated PC12ds (density selected), differed substantially from the original cells. Most notable was a relatively lower content of free [Ca2+]i in the PC12ds cells, as independently assayed by using fluo-3 and indo-1 dyes. In addition, the variant cells exhibited a significantly diminished rate of 45Ca2+ uptake, most likely due to less efficient L-type voltage-dependent calcium (VDC) channels. Addition of several calcium channels agonists and antagonists suggested that PC12ds cells contained relatively more N-type VDC channels, possibly indicating a shift to a neuronal phenotype.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed1992

Identification of two distinct populations of protein kinase C in rat brain membranes.

The regulatory enzyme protein kinase C (PKC) is proposed to be activated on its translocation from the cytosol to the membrane. However, a portion of the native activity is always associated with the membrane fraction. Using a noninvasive procedure to extract this endogenous activity from rat brain membranes, it has been possible to characterize the activity in a partially purified reconstituted system bearing resemblance to the in vivo system. Two subpopulations of membrane-associated PKC were identified and characterized at the level of activation, inhibition, and isozyme immunologic characteristics and chromatographic properties. One peak had properties similar to those of cytosolic PKC, whereas the second population, extracted as protein-lipid complexes, had considerable constitutive activity that could be stimulated further on addition of PKC activators. This latter activity was relatively resistant to staurosporine inhibition and phorbol ester treatment, but it phosphorylated the exogenous PKC substrates, histone 1 and the epidermal growth factor receptor peptide KTRLRR. The constitutive activity was totally dependent on its endogenous associated lipids coextracted by the solubilization procedure. The ratio between these two populations was ontogenetically regulated and modulated by phorbol ester treatment, suggesting that different PKC populations may serve unique functions in the rat brain regulated by the lipid environment. Analyses of the phospholipids extracted in these protein-lipid complexes showed differences in the major classes correlating to age. However, apart from a markedly lower cholesterol content in these complexes, no direct relationship between a specific lipid composition and the amount of constitutive PKC activity was evident.

Animals↗
PubMed1992

Generation of arachidonic acid and diacylglycerol second messengers from polyphosphoinositides in ischemic fetal brain.

Intracerebral administration of [3H]arachidonic acid ([3H]ArA) into 19-20-day-old rat embryos, resulted in a rapid incorporation of label into brain lipids. One hour after injection, 55.6 +/- 8.2, 18.0 +/- 3.4, and 13.7 +/- 1.3% of the total radioactivity was associated with phosphatidylcholine, phosphatidylinositol, and phosphatidylethanolamine, respectively. Approximately 10% of radioactivity was found acylated in neutral lipids of which free ArA comprised only 1.5 +/- 0.2% of the total radioactivity. Complete restriction of the maternal-fetal circulation for < or = 40 min did not affect the rate of [3H]ArA incorporation (t1/2 = 2 min) into fetal brain lipids, suggesting an effective acylation mechanism that proceeds irrespective of the impaired blood flow. After a short restriction period (5 min), the radioactivity in diacylglycerol was elevated by 50%. After a longer restriction period (20 min), the radioactivity in the free fatty acid and diacylglycerol fractions increased to values of 130 and 87%, respectively. Polyphosphoinositides prelabeled with either [3H]ArA or 32P were rapidly degraded after 5 min of ischemia. After 20 min, the decrease in phosphatidylinositol-4-phosphate and phosphatidylinositol-4,5-bisphosphate radioactivity was 47 and 70%, respectively. Double labeling of phospholipids with [14C]palmitic acid and [3H]ArA indicated a preferential loss of [3H]ArA within the polyphosphoinositide species after 20 min, but not after 5 min of ischemia. The specific activity of [14C]palmitate remained unchanged. The current data suggest phospholipase C-mediated diacylglycerol formation at the beginning of the insult followed by a phospholipase A2-mediated ArA liberation at a later time, both enzymes presumably acting preferentially on polyphosphoinositide species.

Animals↗
PubMed1992

Platelet-activating factor stimulates arachidonic acid release and enhances thromboxane B2 production in intact fetal rat brain ex vivo.

The ability of brain preparations from 20-day-old rat fetuses to synthesize eicosanoids in the presence of platelet activating factor (PAF) was investigated. A rise (49%) in thromboxane B2 (TxB2; the stable thromboxane A2 metabolite) was observed after 30 min in the presence of 0.6 microM PAF. Repetitive administration of PAF did not rise TxB2 production above a certain level, suggesting desensitization. 1-O-alkyl, sn-glycero-3-phosphocholine (lyso-PAF) at 0.6 microM had no effect, whereas selective PAF antagonists, i.e., BN52021, BN50739 and BN50727, or indomethacin, a general cyclooxygenase inhibitor, blocked completely TxB2 synthesis. The calcium ionophore A23187 (10 microM) stimulated production of TxB2, prostaglandin E2 and 6-keto-prostaglandin F1 alpha eicosanoids, whereas extracellular calcium deprivation did not impair eicosanoid release. The effects of PAF and A23187 on TxB2 synthesis were not additive and were not dependent on extracellular calcium. Chelation of intracellular Ca++ by Fluo-3/AM reduced production of TxB2 and prostaglandin E2 eicosanoids. Fluo-3/AM also blocked effectively PAF-dependent TxB2 release, indicating that production of TxB2 was almost entirely dependent on free intracellular calcium levels. PAF-dependent changes in brain phospholipids, prelabeled with [3H]arachidonic acid, were examined. One hour after in vivo injection of the isotope, fetal brains were removed and incubated in vitro for 30 min with carbamyl-PAF. Radioactivity in arachidonic acid and diglyceride fractions increased (35% and 30%, respectively), whereas radioactivity in phosphatidylcholine, phosphatidylethanolamine and phosphatidylinositol decreased. BN50726 antagonist abolished the effect of PAF. The radioactivity in poly-phosphoinositides was diminished (30-40% decrease) after PAF addition.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

Reduction of protein kinase C activity in the adult rat brain following transient forebrain ischemia.

Acute forebrain ischemia reduced protein kinase C (PKC) activity in the adult rat cortex, striatum and hippocampus by 60-70% after 20 min ischemia episodes, followed by 48 h of recirculation. Ischemia of 1 min, followed by recirculation, produced a less pronounced but significant decrease in PKC activity. The ischemia-induced decrease of PKC affected both the soluble and the membrane-bound kinase. Alterations of PKC predate neuronal death following ischemia.

Animals↗
PubMed1991

Nerve growth factor mediates monosialoganglioside-induced release of fibronectin and J1/tenascin from C6 glioma cells.

C6 rat glioma cells incubated in serum-free medium with D-[14C]glucosamine secrete, on stimulation with nerve growth factor (NGF) or monosialogangliosides (MSGs), several glycoproteins (Gps), the most prominent of which are a 270-, 220-, and 69-kDa Gp. Several growth factors, hormones, phorbol ester, and disialo- and trisialogangliosides did not stimulate secretion. Western blot analysis of the conditioned medium from C6 cells stimulated with NGF or MSG identified one distinct band of approximately 220 kDa for fibronectin and J1/tenascin, which comigrated. Antiserum to NGF prevented NGF-stimulated release and also blocked MSG-evoked release. The 220-kDa band was labeled after pulse labeling with [35S]methionine in the presence of NGF, and by a 15-min chase period radioactively labeled J1/tenascin could be immunoprecipitated. Tunicamycin drastically inhibited almost completely release of the 220-kDa Gp labeled by D-[14C]glucosamine or [35S]methionine. These results extend the range of neurotrophic properties attributed to NGF to cells of glial origin and suggest that NGF regulates secretion of extracellular matrix proteins. MSG stimulation of fibronectin and J1/tenascin secretion may be mediated by NGF or an NGF-like molecule also secreted by the C6 glioma cells.

Animals↗
PubMed1991

Exogenous GM3 ganglioside stimulates process formation and glycoprotein release by cultured bovine oligodendrocytes.

Isolated adult bovine oligodendrocytes maintained in vitro for 10 days were treated for 1 day with 50 micrograms/ml of GM3 ganglioside (NeuNac alpha 2-3Gal beta 1-4Glc beta 1-1'ceramide) in serum-free culture medium. The treated oligodendrocytes had significantly longer processes with more branching than control cells in the same medium without GM3. The treatment also stimulated the release of a series of 22-100-kDa, [3H]glucosamine-labeled glycoproteins into the culture medium. Treatment of oligodendrocytes maintained in vitro for 50 days with GM3 for 1 day resulted in a thickening of the processes and the appearance of many fine branches on existing processes as well as a similar stimulation of glycoprotein release into the medium.

Animals↗
PubMed1990

Application of high-pressure to subfractionate membrane protein-lipid complexes: a case study of protein kinase C.

Current procedures for solubilization of membrane proteins involve the use of detergents. A procedure using high hydrostatic pressures without detergent has been applied in this study to subfractionate membrane proteins and their endogenously associated lipids. Rat brain membrane preparations were suspended in hypotonic buffer containing the membrane fluidizer benzyl alcohol in a sealed pressure cell and subjected to hydrostatic pressures of up to 1500 atmospheres (approx 22,000 psi) in a French press. Under these conditions, specific membrane proteins including protein kinase C, phospholipase A2, calmodulin-binding proteins, G-proteins, and microtubule-associated proteins all coextracted and were associated to lipid particles, suggesting inherent physical contact. Two populations of membrane-associated protein kinase C were identified according to molecular weight estimations. The first coeluted with the lipid particles composed predominantly of phospholipids, while the second contained much less lipid and was similar to the soluble monomer, i.e., cytosolic protein kinase C. This procedure provides an important technique for selective subfractionation of membrane proteins in their native lipid environment which could be used for structure-function studies.

Animals↗
PubMed1990

Tetanus toxin-induced protein kinase C activation and elevated serotonin levels in the perinatal rat brain.

A single intracerebral injection of tetanus toxin (TeTox) is able to produce a time-dependent translocation of Ca2(+)-phosphatidylserine-dependent protein kinase C (PKC) in close-to-term rat brain. TeTox-triggered translocation of PKC is dose- and time-dependent, can be prevented by tetanus antitoxin, and does not occur upon administration of toxin fragments B and C. TeTox-triggered PKC translocation is accompanied by a time-dependent increase in brain serotonin (5-HT). Increase of brain 5-HT is independent of monoamine oxidase inhibition by pargyline. Phorbol ester and TeTox cause a significant increase in serotonin while H-7, a kinase inhibitor, does not affect serotonin levels but abolishes the effect of TeTox. Gangliosides prevent TeTox-triggered 5-HT increase. The data are consistent with the possibility that TeTox acts effectively on the serotonergic innervation, presumably in conjunction with PKC to cause accumulation of serotonin.

Animals↗
PubMed1990

Preparation of affinity-purified, biotinylated tetanus toxin, and characterization and localization of cell surface binding sites on nerve growth factor-treated PC12 cells.

Biotinylated derivatives of tetanus toxin were prepared and isolated by chromatofocusing and ganglioside-affinity chromatography. Biotinylation was monitored by the appearance of a 210,00 dalton complex upon SDS-polyacrylamide gel electrophoresis in the presence of avidin, and by selective binding to an avidin-Sepharose gel. At molar biotin:toxin ratios from 1:1 to 20:1 only biotinylated derivatives with low toxicity were obtained; these derivatives, however, retained 60-80% of their specific binding affinity for brain synaptosomes. A biotinylated tetanus toxin derivative purified by ganglioside-affinity chromatography was used to identify and localize tetanus toxin binding sites on PC12 cells. Electron microscopic analysis with streptavidin-gold revealed very low levels of tetanus toxin binding sites on the surface of untreated cells, and the appearance of such binding sites during the second week of nerve growth factor-induced differentiation. Examination of micrographs of the differentiated cells indicated that the tetanus toxin binding sites sites are concentrated on the neurites, with relatively few appearing on the cell bodies. Cognate studies using 125I-labeled, affinity-purified tetanus toxin revealed an increase in PC12 binding capacity from about 0.07 nmol/mg protein in untreated cells to 0.8 nmoles/mg protein in cells treated for 14 days with nerve growth factor. Cells treated in suspension for 2-3 weeks with nerve growth factor do not express tetanus toxin binding sites; upon plating, these cells required one week for the appearance of binding sites, although neurites grew much more rapidly from these "primed" cells. The high binding capacity of these tetanus toxin sites, as well as their sensitivity to neuraminidase, is indicative of a polysialoganglioside structure. The advantages of biotinylated tetanus toxin derivatives are discussed and the significance of nerve growth factor-differentiated PC12 cells grown as monolayers as a model for the study of the development, localization, and function of neuraminidase-sensitive tetanus toxin binding sites is presented.

Adrenal Gland Neoplasms↗
PubMed1990

Cyclic GMP alterations in fetal rat cerebrum after global intrauterine ischemia: role of guanylate cyclase phosphorylation.

Changes in the levels of cyclic AMP (cAMP) and cyclic GMP (cGMP) have been measured in brains of 20-day-old rat fetuses exposed to global intrauterine ischemia. Ischemia of different duration (0.5-30 minutes) did not alter the level of cAMP. In contrast, cGMP levels increased as a result of ischemia. This increase was seen even after a short period of ischemia (less than 5 minutes) and was maximal after 5 minutes, where a threefold increase could be observed. This stimulation was transient: after 30 min of ischemia, cGMP returned to the control level. Accumulation of cGMP can be related to the activation of guanylate cyclase, the activity of which is doubled after 15 minutes of ischemia. Immunoprecipitation of guanylate cyclase after in vivo labeling of the fetal brain with 32Pi revealed a threefold increase in the phosphorylation of the enzyme after 15 minutes of ischemia. The possible role of these modifications in cGMP metabolism during the course of ischemia is discussed.

Animals↗
PubMed1990

Thromboxane and prostacyclin levels in fetal rabbit brain and placenta after intrauterine partial ischemic episodes.

The appearance of arachidonic acid (AA) oxidation products in fetal rabbit brain and placenta under normal or partial short-term ischemic episodes induced by placental blood vessel restriction was examined. Intracerebral administration of [3H]AA into close-to-term rabbit fetuses gave rise to radioactively labeled prostaglandin (PG) E2, thromboxane B2, and 6-keto-PGF1 alpha metabolites as detected by HPLC analysis. A significant increase of 20-30% of [3H]AA precursor into eicosanoids was detected in brain of fetuses after 2-h restriction. The thromboxane B2 and 6-keto-PGF1 alpha levels were determined by radioimmunoassay technique over a period of 48 h following ischemic episodes. Thromboxane B2 content in affected animals was higher by five- and twofold at 3 h over control fetal brain and placental tissue values, respectively, and remained significantly higher for 24 h. 6-Keto-PGF1 alpha levels reached a peak value that was greater by 2.5- and 1.5-fold at 6 h for the ischemic brain and placental tissue, respectively, compared with control fetuses. PGE2 levels were less affected, attaining a maximum of 1.9- and 1.1-fold in brain and placenta correspondingly. The thromboxane/prostacyclin ratio reached a maximum in the brain after approximately 3 h, while that in the placenta continued to rise even after 20 h. Persisting high levels of thromboxane are indicative of cerebral vasoconstriction and may suggest possible damaging effects.

Animals↗
PubMed1990

Gangliosides prevent ischemia-induced down-regulation of protein kinase C in fetal rat brain.

Complete obstruction of the maternal blood flow to fetal rats at 20 days of gestation for a period of 10 min causes a significant shift of approximately 22% in protein kinase C (PKC) activity from a cytosolic to a membrane-bound form in the fetal brain. This translocation can be entirely reversed without losses in activity by a single intraperitoneal injection into the gravid rat of either a mixture of disialo- and trisialoganglioside [polysialoganglioside (PSG)] or by GM1 (50 mg/kg of body weight) given 3 h before onset of the ischemic episode. Cessation of blood flow for 15 min followed by a reperfusion period of 24 h results in a 47% loss in total PKC activity. This down-regulation can be almost entirely prevented upon intraperitoneal administration of GM1 3 h before, but also during and even 90 min after the onset of ischemia. The PSG mixture is also effective, particularly when given 3 h before the insult. Down-regulation of PKC is accompanied by an increase in a Ca2(+)-phosphatidylserine-independent kinase [protein kinase M (PKM)] activity, which rises from 30 pmol/min/mg of protein in control animals to a maximal value of 83.1 pmol/min/mg of protein after 15 min of ischemia and 6 h of reperfusion. By 24 h, PKM activity is 46.8 pmol/min/mg of protein. Administration of GM1 blocks completely the appearance of PKM, a result suggesting that PKC down-regulation and PKM activity elevation are intimately associated events and that both are regulated by GM1 ganglioside.

Animals↗