Biomedical subjects

E Yavin

Publications and source records attributed to E Yavin.

At least 73 records · Page 4Linked to original sources

PubMed1990

In vivo translocation and down-regulation of protein kinase C following intraventricular administration of tetanus toxin.

A single intraventricular injection into adult rats of 100 mouse lethal doses of tetanus toxin (TeTox) produces a marked intracellular redistribution of Ca2+/phosphatidylserine (PtdSer)-dependent protein kinase C (PKC) activity. Changes are particularly pronounced in hypothalamus, hippocampus, and spinal cord structures. Translocation of PKC from the inactive cytosolic compartment to a membrane-bound active form is followed by a time-dependent reduction in both total activity and enzyme protein. The down-regulation of PKC activity in the hypothalamus is accompanied by a marked increase in a Ca2+/PtdSer-independent kinase activity, predominantly in the cytosolic fraction. Our data identify PKC as a possible indirect target for TeTox and suggest that down-regulation of the enzyme may provide a clue for tetanus neurotoxicity.

Animals↗

PubMed1989

Phospholipid-derived choline intermediates and acetylcholine synthesis in mouse brain synaptosomes.

Endogenous free choline levels and acetylcholine (ACh) synthesis in nerve terminals were investigated using cerebral cortical synaptosomes of C57BL/6 mice. Endogenous choline was produced at a rate ten-fold faster than ACh to provide levels adequate for the formation of the latter. The combined pool size of the water-soluble intermediates derived from phosphatidylcholine (PhC), such as glycerophosphorylcholine (GpCh) and phosphorylcholine (PCh), increased significantly during the first 10-15 min of incubation and was always higher than that of free choline. These results most likely indicate an effective degradation of PhC by the combined action of phospholipase A2/lysophospholipase, as well as by phospholipase C in synaptosomes. ACh synthesis proceeded at a constant rate in the presence or absence of exogenous free choline (0-10 microM) and was almost entirely abolished in the presence of 10(-6) M hemicholinium-3. These results suggest that ACh is effectively synthesized by free choline generated in synaptosomes by a coupling mechanism involving the high-affinity choline uptake system. No changes in the production rates of choline and ACh were observed between adult and aged mice.

Acetylcholine↗

PubMed1989

Periodate-modified gangliosides enhance surface binding of tetanus toxin to PC12 pheochromocytoma cells.

The interaction of 125I-labeled tetanus toxin with PC12 pheochromocytoma cells in monolayer cultures has been examined. Under regular growth conditions, the PC12 cells bind 125I-tetanus toxin to a limited degree compared with dissociated cerebral neuron cultures. After exposure to nerve growth factor for 2 days in low serum-containing media with growth factor supplements, binding of toxin increases over twofold compared with untreated PC12 cells. Binding can also be enhanced (greater than 2.5-fold) after treatment of cells with 2 mM sodium metaperiodate for 20 min. Dissociated cerebral neurons but not fibroblasts in cell culture bind more toxin after periodate treatment. The effect of periodate can be abolished by 5 mM sodium borohydride. A ganglioside isolated from periodate-treated PC12 cells and tentatively identified as GT1b [(N-acetylneuraminyl)galactosyl-N-acetylgalactosaminyl(N- acetylneuraminyl-N-acetylneuraminyl)-galactosyl-glucosylceramide] binds 125I-tetanus toxin on silica gel chromatoplates and on nitrocellulose paper. There are no indications to suggest binding to a polypeptide from treated cells after polyacrylamide gel electrophoresis. Cells artificially supplemented with GT1b and subsequently treated with periodate effectively bind the toxin. The data suggest that modified sialyl groups linked to gangliosides, and not to proteins, are preferential targets for tetanus toxin.

Borohydrides↗

PubMed1989

Ischemia stress and arachidonic acid metabolites in the fetal brain.

Hypoxic-ischemic insults caused by placental insufficiency in perinatal life are today considered a major cause for neuronal injury and impaired postnatal development. A major consequence of placental insufficiency and ischemia is the change in metabolism of arachidonic acid and its oxidation products. A burst of postischemic production of prostaglandins, unequivocally shown in many systems, is documented in the fetal rabbit brain as well as in placenta tissue soon after vascular restriction. PGE2, a most abundant prostaglandin of the fetal brain, is particularly elevated. Similarly, thromboxane B2 and 6-keto PGF1 alpha, the stable metabolites of thromboxane A2 and prostacyclin, are both increased over the control values. However, after 48 h of restriction, the levels of these eicosanoids are restored to near-normal values. The metabolic machinery responsible for the conversion of arachidonic acid into eicosanoids in brain and placenta tissues appears to be impaired following a period of placental insufficiency. This inhibition can be accounted for by excessive production of eicosanoids and also by formation of an endogenous inhibitor or free radicals. Studies are in progress to test these possibilities.

Animals↗

PubMed1988

Protein kinase C alterations in the fetal rat brain after global ischemia.

Marked changes in the intracellular localization of brain protein kinase C are evident after global ischemia generated by the restriction of the placental blood flow in the near-term rat embryo. A rapid (5 min) ischemia-dependent translocation of the enzyme from the cytosol to the particulate membrane fraction, which is completely reversible upon reperfusion, is observed. After 30 min of ischemia, substantial losses in protein kinase C activity and content as measured by [3H]phorbol dibutyrate binding are apparent. This is accompanied by a marked increase of a Ca2+-phosphatidylserine-independent kinase activity, already evident after 5 min of ischemia. By 15 or 30 min the total activity of the latter enzyme is equally distributed between the particulate and the cytosol fractions and is more than 3-fold higher in ischemic in comparison to naive animals. Activation and possible deregulation of protein kinase C are proposed to represent an initial step in the pathophysiology of brain ischemia.

Animals↗

PubMed1988

Acute uteroplacental ischemic embryo: lactic acid accumulation and prostaglandin production in the fetal rat brain.

A new experimental model for studying the effects of acute ischemia on brain development in the near-term fetal rat has been devised. Ischemic conditions are achieved by complete clamping of blood vessels branching from the uterine vasculature into each individual fetus for designated times followed by removal of the clamps to permit reperfusion. Accumulation of lactic acid in the fetal brain depends on the length of the restriction period, reaching a plateau level of 29 mumol/g tissue at about 30 min. It also depends on the reperfusion time. Thus after a period of 15 min of restriction lactate levels show an increase over the next 30-min reperfusion to a value of 25.5 mumol/g followed by a rapid decrease to normal values by 3 h of reperfusion. Restriction of 15 min followed by reperfusion of 45 min causes an elevation of prostaglandin E2 (PGE2) level from 12.4 +/- 0.86 ng/g to 21.1 +/- 0.6 ng/g (p less than 0.001). This elevation in PGE2 level is less apparent after 20 min of restriction. No effects are seen on the level of PGF2 alpha. Both PGE2 and PGF2 alpha accumulate in vitro in a time-dependent manner by brain particulate fraction. In vitro synthesis of both PGE2 and PGF2 alpha is inhibited by indomethacin (100% and 68%, respectively) and AA861 (94% and 76%, respectively). BW755c and nordihydroguaiaretic acid do not affect PGE2 formation but enhance PGF2 alpha production by 112% and 152%, respectively. Particulate fractions from restricted brain produce less PGF2 alpha than control brains (6.38 +/- 1.62 versus 11.43 +/- 2.2, p less than 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)

4,5-Dihydro-1-(3-(trifluoromethyl)phenyl)-1H-pyraz↗

PubMed1987

Molecular interactions between micellar polysialogangliosides and affinity-purified tetanotoxins in aqueous solution.

Two-affinity purified tetanotoxin forms, TeToA and TeToB, with different affinities for gangliosides were characterized by analytical ultracentrifuge, circular dichroism (CD), and amino acid composition. Both toxin forms share a common sedimentation coefficient of about 6-7 S and similar alpha-helicity values, but they vary in amino acid composition. Incubation of TeToB with micellar polysialogangliosides results in formation of high (21-24 S) and medium (13-15 S) size toxin-micellar ganglioside aggregates as revealed by analytical ultracentrifuge technique. At TeToB/[N-acetylneuraminyl]-galactosyl-N-acetylgalactosaminyl- [N-acetylneuraminyl-N-acetylneuraminyl]-galactosylglucosylceramide (GT1b) molar ratios of greater than 26, high molecular weight aggregates (Mr greater than or equal to 700,000) which contain between 3 and 5 toxin monomers are formed, whereas at molar ratios less than 15, about 1-2 monomers are present. TeToA does not form aggregates in the presence of gangliosides. A marked increase in the alpha-helix from about 20 to 39% is apparent in the CD spectrum of TeToB after interaction with ganglioside mixture (G1b). Cerebrosides, sulfatides, sphingomyelin, and phosphatidylserine also increase the alpha-helix, presumably because of an overall effect of lipids on the protein. TeToA and fragment B but not C also undergo similar changes in the presence of G1b, suggesting that the effect of ganglioside is not specific. The polarity of the CD spectra of a number of gangliosides is shifted from a negative to a positive value after interaction with tetanotoxin. The data are consistent with the interpretation of a discrete hydrophobic domain on the toxin heavy chain which interacts with micellar gangliosides to form macromolecular complexes.

Amino Acids↗

PubMed1987

Selective enhancement of tubulin gene expression and increase in oligo(dT)-bound RNA in the rat brain after nigrostriatal pathway unilateral lesion and treatment with ganglioside.

Partial hemitransection of the rat nigrostriatal pathway has been applied to study changes in the expression of tubulin and actin cytoskeletal genes. RNA was isolated from ipsilateral and contralateral structures of substantia nigra and striatum tissues of lesioned or sham-operated animals by a combined proteinase K and oligo(dT)-cellulose affinity purification procedure. A significant increase in the oligo(dT)-cellulose-eluted RNA was observed in the lesioned substantia nigra and striatal tissues compared to naive controls. By 5 days, the RNA content of the lesioned nigra reached a value of 96-114.5 micrograms/mg DNA compared to 45.7-52.9 micrograms/mg DNA. Administration of GM1 gangliosides at daily intervals resulted by day 5 in a further increase (131.8-141.5 micrograms/mg DNA) in the RNA content of the lesioned but also of the sham-operated (81.9-97.8 micrograms/mg DNA) animals. By 18 hr, the lesioned nigra exhibited a four-fold increase in tubulin messenger RNA (mRNAtub) gene expression in comparison to the sham-operated animals. The substantia nigra tissue of GM1-treated animals exhibited a hybridization value for mRNAtub twice as high compared to GM1-untreated, lesioned animals. The effect of GM1 appeared selective for alpha-tubulin and beta-tubulin compared to actin transcript. The latter was also elevated in the lesioned nigra tissue above the naive or sham-operated animals. The data suggest that, after injury, cytoskeletal RNA transcripts are elevated and that GM1 may play a role in the regulation of their steady-state levels.

Animals↗

PubMed1987

Arachidonic acid oxidation by brain and placenta preparations from normal and placental insufficient fetal rabbit.

Cytosolic (100,000 g) fractions of fetal rabbit brain and placenta tissue convert [1-14C]arachidonic acid into several oxidation products identified with the lipoxygenase [12-hydroxyeicosatetraenoic acid (12-HETE) and 15-HETE] and cyclooxygenase [prostaglandin E2 (PGE2)] pathways. Formation of 12-HETE and 15-HETE by fetal brain is time-dependent, reaching a plateau after 40 min and is linear with protein concentration. An apparent affinity constant of 0.06 mM and a Vmax of 0.1 mumol/h/g wet weight are presumably responsible for the excessive accumulation of 12-HETE and 15-HETE in comparison to PGE2 (Km = 0.5 mM). The latter is synthesized by the placenta particulate fraction but almost exclusively by the brain cytosol. Compared to brain, the activity of the placenta tissue is exceedingly higher and in addition to 12-HETE and 15-HETE there is a substantial formation of 12-L-hydroxyheptadecatrienic acid. Formation of 12-HETE and 15-HETE at 21 days is as effective as at 31 days gestation and is strongly inhibited by nordihydroguaiaretic acid (93%), BW755c (99%), and AA861 (84%) but not by indomethacin. Placenta and brain tissues of intrauterine growth retarded fetuses after ligation of placental blood vessels fail to convert arachidonic acid into other eicosanoids. Loss of enzymatic activity also observed in normal tissue after prolonged storage cannot be restored by the addition of several SH agents, ascorbate, or ferric iron.

12-Hydroxy-5,8,10,14-eicosatetraenoic Acid↗

PubMed1986

Affinity-purified tetanus neurotoxin interaction with synaptic membranes: properties of a protease-sensitive receptor component.

The pharmacokinetic interaction of an affinity-purified 125I-labeled tetanotoxin fraction with guinea pig brain synaptosomal preparations was investigated. Binding of tetanotoxin was time- and temperature-dependent, was proportional to protein concentration, and was saturable at about 8 X 10(-9) M as estimated by a solid-surface binding assay. Binding was optimal at pH 6.5 under low ionic strength buffer and was almost entirely blocked by gangliosides or antitoxin. In analogy to intact nerve cells, binding of toxin to membranes resulted in a tight association operationally defined as sequestration. Binding and sequestration were abolished after membrane pretreatment with sialidase. The enzyme could not dissociate the membrane-bound toxin formed at 4 or 37 degrees C under low ionic strength conditions, which is in part compatible with internalization as defined in nerve cell cultures. In the latter system the toxin could be removed at 4 degrees C but not at 37 degrees C. Binding was significantly reduced upon pretreatment of guinea pig brain membranes by a variety of hydrolytic enzymes. Trypsin and chymotrypsin inhibited binding between 55% and 68% while bacterial protease abolished it by 91-95%. The effect was species-specific as it was not seen in rat or bovine synaptosomes. Collagenase and hyaluronidase had little or no inhibitory effect when applied to synaptosomes (27% and 9%) but inhibited binding to synaptic vesicles by 56% and 49%, respectively. Phospholipases A2 and C caused 42-43% inhibition of binding in vesicles and less than 22% in synaptosomes.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1986

Tetanus toxin receptors on nerve cells contain a trypsin-sensitive component.

Cerebral neurons in monolayer cultures, subjected to 25 micrograms/ml trypsin, lose after 10 min about 43.5% and 40.5% of the ability to bind 125I-labeled tetanotoxin as measured at 0-4 degrees C and 37 degrees C respectively. These losses are maximal by 30 min and can be prevented by 1.5 mg/ml soybean trypsin inhibitor. Chymotrypsin but not collagenase or hyaluronidase is also effective in reducing binding of toxin to cells. The trypsin-insensitive toxin-binding activity can be further eliminated by treatment with sialidase or by cell extraction with methanol. Fixation of cells with 3.5% paraformaldehyde or 2% glutaraldehyde also results in a marked decrease of 52.4% and 25% respectively in the toxin-cell association. Methanol or sialidase but not trypsin removes the remaining binding activity. About one-third of the lipid-linked and protein-linked sialic acid is removed after sialidase treatment whereas 1% and 9.4% respectively are removed after trypsin treatment. The data are consistent with the possibility that, in addition to a sialic acid component, binding of tetanotoxin to nerve cells is facilitated by a trypsin-removable and formaldehyde-inactivated component. There was no evidence for a polypeptide to substitute gangliosides as receptors for tetanotoxin. On the contrary, solubility in organic solvents and interaction of the extracted products with labeled toxin remain the major proof that gangliosides are the putative receptors for tetanotoxin.

Animals↗

PubMed1986

Muscarinic binding sites in newborn brain from intrauterine growth-retarded rabbits.

Intrauterine growth retardation (IUGR) was induced in rabbits by means of vascular ligation in utero 4-6 days prior to birth. The vascular-restricted living newborns and controls from the unligated uterine horns were killed at birth and the level of the muscarinic cholinergic receptors in cerebral and cerebellar homogenates measured using the potent antagonist 3H-QNB. No significant difference in the binding sites of the cerebellum and the cerebral cortex was found between restricted and control animals. It is speculated that the ineffectiveness of the vascular insult to affect specifically muscarinic binding sites could partially be explained by the specificity of the receptors and the timing of the ischemic insult in relation to the stage of the developing brain. Other cholinergic markers should also be assessed.

Animals↗

PubMed1986

Nerve growth factor and gangliosides stimulate the release of glycoproteins from PC12 pheochromocytoma cells.

PC12 pheochromocytoma cells in monolayer cultures secrete increased amounts of glycoproteins into the medium following the addition of nerve growth factor (NGF) or of brain gangliosides. After a 48-h incubation with 50 ng/ml NGF there is approximately a twofold increase in the total [14C]glucosamine-labeled, ethanol-precipitable cellular material released into the medium. Between 30 and 50% of the radioactivity is associated with a glycoprotein (Gpl) of molecular weight of 52,000; the remaining radioactivity is distributed between five and six major bands. Only a small amount (10%) is associated with a glycoprotein of Mr greater than 200,000 which might correspond to the NGF-induced large external glycoprotein. A substantial increase in the release of the glycoproteins is also seen on the addition of a variety of gangliosides including asialo GMl. This increase is independent of the presence of NGF. GMl and GDlb/GTlb but not GDla stimulate release above the levels seen in the presence of NGF. Addition of GDla (2 micrograms/ml) enhances selectively the release of various glycoproteins between 2.6- and 8-fold. The pattern of glycoprotein secretion is similar to that seen with NGF, although Gp2 (Mr 78,000) is more abundant. Stimulation of release by GDla is not accompanied by neurite outgrowth, suggesting that the glycoproteins are not directly associated with neuritogenesis. The release of these glycoproteins following the addition of NGF or gangliosides may relate to the neurotrophic properties that these two entirely different ligands exert on PC12 cells.

Adrenal Gland Neoplasms↗

PubMed1985

Calcium-independent phosphatidylinositol response in gonadotropin-releasing-hormone-stimulated pituitary cells.

This paper describes the effect of gonadotropin-releasing hormone (GnRH, gonadoliberin) on phospholipid metabolism in cultured rat pituitary cells. The cells were incubated with [32P]Pi to label endogenous phospholipids (10-60 min) and then stimulated with GnRH for up to 60 min. Cellular phospholipids were separated by two-dimensional t.l.c. and the radioactivity was determined. Phosphatidylinositol (PI), a minor constituent of cellular phospholipids (7.7%), was the major labelled phospholipid, accounting for 45% of the total radioactivity, at early periods after pulse labelling. On the other hand, phosphatidylcholine, the major cellular phospholipid (37%), was labelled only to 32% of the total radioactivity. The remaining label was distributed among phosphatidylethanolamine (4.2%), cardiolipin (3.4%), phosphatidic acid (PA, 2.5%), and phosphatidylserine (1.8%). GnRH doubled 32P labelling of PA and PI significantly at 1 and 5 min of incubation respectively in the presence or absence of extracellular Ca2+. Labelling of other phospholipids was not affected by GnRH treatment. The half-maximal stimulating dose (ED50) for PI labelling and lutropin release was 0.75 nM and 0.5 nM respectively, and the stimulatory effect was blocked by the potent GnRH antagonist [D-Glp1,pClPhe2,D-Trp3,6]GnRH. GnRH-stimulated PA and PI labelling could not be demonstrated after 1 and 45 min of incubation respectively, or when the prelabelling was conducted for 60 min rather than 10 min. These results suggest heterogeneous compartmentalization of gonadotroph PA and PI pools and that increased PI turnover might be a transducing signal for Ca2+ gating that follows gonadotroph GnRH-receptor activation.

Animals↗

PubMed1985

Tetanus toxin interaction with human erythrocytes. I. Properties of polysialoganglioside association with the cell surface.

Human erythrocytes in suspension acquire gangliosides containing di- and trisialosyl residues added to the maintenance medium. This is reflected in the increased cell-associated sialic acid content and ability to bind 125I-labeled tetanus toxin. A salt-sensitive and a salt-insensitive ganglioside-mediated toxin-cell surface association is detected which is reduced after sialidase treatment of ganglioside-supplemented cells. The salt-insensitive ganglioside-cell association is saturable after 2 h incubation in 0.3 M mannitol buffer and has an optimum at pH 5. The association process is higher at 37 degrees C than at 4 degrees C, depends on cell density, and is considerably higher in metabolically active cells compared to lysed cells. Pretreatment of cells with trypsin decreases the salt-resistant toxin association with ganglioside-supplemented cells. In contrast, glutaraldehyde-fixed cells treated with trypsin and supplemented with gangliosides bind more toxin which is insensitive to salt. Ganglioside-mediated tetanus toxin binding to the intact erythrocyte membrane can be utilized as a model system for studying the role of glycolipids in membrane function.

Cell Count↗

PubMed1985

Tetanus toxin interaction with human erythrocytes. II. Kinetic properties of toxin association and evidence for a ganglioside-toxin macromolecular complex formation.

The properties of tetanus toxin interaction with human erythrocytes supplemented with disialo- and trisialo-gangliosides have been investigated. Binding of toxin is linear with time for 1 h and is 3-4-fold higher at 37 degrees C than at 4 degrees C during incubation of long duration. It exhibits saturation at toxin concentrations between 0.1 and 1 microgram/ml; however, it is nonsaturable between 1 and up to 50 micrograms/ml. It is effectively prevented by free gangliosides and antibodies or by pretreatment with sialidase but is unaffected by a number of closely related ligands including toxoid and toxin fragments. NaCl (1 M) removes a great portion (86%) of cell-associated toxin while Triton X-100 extracts an additional fraction (30%) of the salt-resistant cell-bound toxin. The residual sequestred toxin after detergent extraction is sensitive to proteolytic degradation. The trypsin-stable fraction (1.5%) is biotoxic and may be indicative of internalization of toxin. A macromolecular complex of about 700 kDa containing toxin and gangliosides has been isolated and characterized by Sephacryl S-300 gel permeation chromatography, SDS-gel electrophoresis, immunoprecipitability and biotoxicity. This complex is obtained only in ganglioside-supplemented cells and not when free 3H-labeled GD1b is reacted with 125I-labeled toxin in solution in the absence of cells. The hydrophobicity properties acquired as a result of ganglioside-toxin interaction, presumably at the cell surface, suggest a conformational change of the toxin which may enable its penetration into the bilayer.

Binding, Competitive↗

PubMed1985

Brain: body ratio and conceptional age in vascular-induced intrauterine growth retarded rabbits.

Vascular induced intrauterine growth retardation (IUGR) was achieved by total ligation of approximately 30% of the placental vessels to half the fetuses in the last third of gestation in pregnant rabbits. A correlation between brain weight, body weight and head circumference was established in fetuses and rabbit pups in the perinatal period. The brain:body ratio in restricted IUGR animals was significantly higher than their homologous normal controls. A cephalization index based on the brain:body ratio is proposed to assess adverse effects on brain maturity in the presence of IUGR induced by placental insufficiency.

Animals↗

PubMed1985

The cephalization index: a screening device for brain maturity and vulnerability in normal and intrauterine growth retarded newborns.

Predictive estimates of future neurological maldevelopment as a result of vascular induced intrauterine injury are based on the assumption that the body is more affected than the brain resulting in asymmetrical intrauterine growth retarded (IUGR) newborns. The higher the brain:body ratio, the more severe the IUGR process and the greater the risk for the brain to be affected. This prompted us to study in human newborns, a cephalization index based on the ratio of head circumference to body weight to express the degree of brain maturity and possible vulnerability in relation to gestational age. The newborn cephalization index was correlated with neurodevelopment. A trend could be delineated; in the later gestational age, the higher the cephalization index reflecting a greater degree of brain vulnerability, the more severe the clinical pathology; especially the likelihood of cerebral palsy and severe psychomotor retardation. The cephalization index may serve as an additional screening device for high risk intrauterine growth retarded newborns.

Body Weight↗