Calf Brain Research Articles

Subfractions of membranes from calf brain synaptosomes obtained and studied by liquid—liquid partitioning

Synaptosomes isolated from calf brain cortex were lysed and fragmented by Yeda press treatment. The obtained membranes have previously been fractionated in a counter-current distribution process using a liquid—liquid two-phase system consisting of water, dextran, Ficoll and poly(ethylene glycol) [ J. Chromatogr., 358 (1986) 147]. Using the fact that there are discrete membrane populations, a rapid preparative method for isolation of the two main fractions is presented in the present work, as well as a subfractionation of one of them using liquid—liquid extraction with dextran-bound Procion yellow HE-3G. The content of several membrane constituents, i.e. protein, acetylcholinesterase, succinate dehydrogenase and ATPase, as well as opiate binding, were determined for the three fractions. Counter-current distribution of the fractions elucidates their heterogeneity and the effectiveness of the purification.

High molecular weight glycosylated protease in calf brain. Purification and partial characterization.

A high molecular weight protease has been purified to homogeneity from calf brain cytosol. The purification procedure involves ammonium sulfate fractionation of the cytosol followed by chromatography on DEAE-Sephacel, hydroxylapatite, concanavalin A-Sepharose 4B and Sephacryl S-300. The molecular weight of the native protease was estimated to be Mr = 465,000 by high pressure liquid chromatography. It is composed of a closely moving doublet of Mr = 165,000 and 155,000, as determined on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It degrades [methyl-14C] alpha-casein with a broad pH optimum of 6.8-8.5. [methyl-14C]bovine serum albumin and 125I-bovine serum albumin are hydrolyzed to the same extent as [methyl-14C]alpha-casein, whereas [methyl-14C]methemoglobin is hydrolyzed to half the extent of [methyl-14C] alpha-casein. Divalent cations, nucleotides, and known protease inhibitors (phenylmethylsulfonyl fluoride, p-chloromercuribenzoate, iodoacetic acid, N-ethylmaleimide, leupeptin, antipain, pepstatin, and hemin) have no effect on the activity of the protease. The protease is glycosylated and appears to aggregate readily. Aggregation may be reversed by treating the protease with certain organic solvents. The protease seems to maintain full activity after heat treatment. Electron microscopic data reveals a spherical structure of 20-nm diameter.

Differential effects of dietary fatty acids on fatty acid composition of phosphotidylinositol in calf tissues

Fatty acid compositions of phosphatidylinositol (PI) in calf liver, heart, muscle, and brain were measured, as affected by dietary treatments. High Cu intake (1000 ppm) decreased 18:0 in liver PI and increased 18:1 n-9 and 18:2 n-6 in liver and heart; the reversed changes were observed in skeletal muscle PI. Additional high Zn intake (1000 ppm) inhibited the high Cu effects. Feeding an essential fatty acid (EFA) deficient diet markedly decreased 18:2 n-6, 20:3 n-6, and 20:4 n-6, and increased 18:1 and 20:3 n-9 in liver and heart PI. A high intake of 18:2 n-6 (corn oil) increased 18:2 n-6, but decreased 20:4 n-6, in liver, heart and muscle PI. Dietary fish oil concentrate (high in eicosapentaenoic acid) increased 20:5 n-3 in liver, heart, and muscle PI. Fatty acid composition of brain PI was resistant to different dietary regimens. Dietary imposed changes in fatty acid composition of PI in liver, heart, and muscle may have important implications in transmembrane signalling, eicosanoid production and other functions attributed to PI in tissues.

Kinetics of dissociation of the tubulin-colchicine complex. Complete reaction scheme and comparison to thermodynamic measurements.

The slow dissociation reaction of the tubulin-colchicine complex has been characterized in purified calf brain tubulin and microtubule protein preparations, using [3H]colchicine and fluorometric measurements. It fits to a single exponential phase, within the accuracy of these measurements. The dissociation is a kinetically unfavorable reaction, with activation energy values of 114 +/- 10 and 94 +/- 10 kJ mol-1 (purified tubulin and microtubule protein, respectively). The kinetic scheme previously proposed for the tubulin-colchicine association (Lambeir, A., and Engelborghs, Y. (1981) J. Biol. Chem. 256, 3279-3282) is: T + C K1 in equilibrium TC k2 in equilibrium k-2 (TC)' where step 1 is a fast reversible binding and step 2 is a slow conformational change, whose backward rate constant (k-2) was neglected for the association study. This kinetic scheme has now been completed to include the measurements of the rate-limiting dissociation step (k-2), and of the purified calf brain tubulin preparation. The overall binding standard free energy change, calculated from the kinetic measurements, is -42.0 +/- 0.1 kJ mol-1 (fast phase of binding in 10 mM sodium phosphate buffer, 0.1 mM GTP, pH 7.0, at 37 degrees C). The binding is exothermic and the calculated enthalpy change is -26 +/- 13 kJ mol-1, which coincides with the recently determined calorimetric enthalpy value, -21 +/- 2 kJ mol-1 (Menendez, M., Laynez, J., Medrano, F. J., and Andreu, J. M. (1989) J. Biol. Chem. 264, 16367-16371), suggesting that the kinetic scheme and measurements are essentially correct.

Transport of drugs across the blood-brain barrier

The blood-brain barrier prevents an indifferent medicine existing in the blood to enter also in the brain. This barrier has got an anatomical base: it is first consisting in a cerebrovascular layer of endothelial capillary vessels of the peripheral tissue. It is moreover covered by outgrowths of the flial cells, which are called astrocytes. There are, for that reason, important limits to a size of molecules which can reach the cerebral tissue through a paracellular way (through what is called in English "tight-junctions"). Most medicines must use the transcellular way. Lipophily is necessary to follow that way. Year after year, it appeared, thanks to a comparative study of the substances, that there exists--grosso modo--a positive correlation between the lipophilic level and the permeation-level of a substance in the cerebral tissue. There are, however, several exceptions: it is so that hydrophilic substances, possessing an important nourishing function (such as glucosis, amino-acids) seem to penetrate much more easily than we could expect when we consider their physicochemical characteristics. This is the result of the fact that there exist specifical transport-mechanisms for these substances at the level of the endothelial cell-membranes, allowing the penetration of such substances. There exist, on the contrary, lipophilic components that penetrate the cerebral tissue much less strongly than we should expect. This happens because there also exist pumping-mechanisms at the level of the hemato-encephalic barrier. The concerning substance, which was recently discovered is the "glycoprotein P", which is also responsible for the "multi-drug-resistance" and for the resistance of tumors to cytostatics. This phenomenon relies on a very efficient pumping of substances which have penetrated cells in which this protein expressed itself in the membranous structure. In order to obtain a better understanding of the function of the hemato-encephalic barrier, comprising the transport of medicines, it is most important to have reliable experimental models. It is to that aim that, during former years, the technique of cultivating endothelial cerebrovascular cells was developed. These cells are isolated from brains of calves or rats and, subsequently, cultivated on a laboratory medium; about a week later, they have grown a single and confluent layer. This layer represents a kint of "hemato-encephalic barrier" in vitro, which allows us to study the transfer of substances through the layer and thus also the details concerning the transport mechanisms, as well as the factors influencing the permeability of the cells-layer (for instance the inflammatory stimuli). Concerning the "in vivo" research, the technique of intracerebral microdialysis in lab-animals proved to be very promising. In order to effect this microdialysis, a semipermeable microcannula is introduced in the brain tissue, across which an iso-osmotic liquid is being injected continuously. The substances staying in the interstitial liquid of the cerebral tissue will diffuse under the influence of a concentration gradient, into the dialysing liquid and they will also be ready to be analysed. Thanks to this technique, it is possible to follow, in the same animal, the evolution of the concentration in the brain of a substance which has, for instance been injected in a peripheral region. In this way, we obtain, indirectly and in vivo, informations about the functioning-process of the "hemato-encephalic barrier". We can, moreover, effect measures on a specific spot, for instance in tumoral brain tissue: this allows us to study the influence of specific transport-mechanisms. These rather recent techniques, as well in vitro as in vivo, will allow us, in consequence, to increase, during the next years, our understanding of the way the hemato-encephalic barrier functions as to the transfer of medicines towards the central nervous system. This understanding may lead us to new strategies allowing

Heterogeneity of synaptosomal membrane preparations from different regions of calf brain studied by partitioning and counter-current distribution

1. 1. Membranes obtained by lysis and Yeda-press treatment of synaptosomes (nerve endings) from cortex, caudateus nucleus, and hippocampal region of calf brain have been studied by partitioning within a liquid-liquid aqueous two-phase system consisting of water, dextran, Ficoll, and poly(ethylene glycol). 2. 2. The partitioning of membranes was sensitive to the presence of a dextran-bound dye, Procion yellow HE-3G, in the lower phase. 3. 3. The two-phase system was used for counter-current distribution to study the heterogeneity of the synaptic membranes from the three regions of the brain and to separate the membranes into fractions. 4. 4. The obtained counter-current distribution profiles strongly depended on the region of the brain from which the membranes were isolated. 5. 5. The membrane fractions obtained showed marked differences in their SDS electrophoresis pattern.

Isolation and heterologous expression of a cDNA encoding bovine inositol polyphosphate 1-phosphatase.

Inositol polyphosphate 1-phosphatase, an enzyme of the phosphatidylinositol signaling pathway, catalyzes the hydrolysis of the 1-position phosphate from inositol 1,3,4-trisphosphate and inositol 1,4-bisphosphate. The protein was isolated from calf brain and digested with trypsin or CNBr, and the amino acid sequence of several peptides was determined. Degenerate oligonucleotide primers were designed from amino acid sequence and used to synthesize an 80-base-pair (bp) fragment by the polymerase chain reaction. This product was used to isolate a 1.6-kbp cDNA with an open reading frame of 400 amino acids, 185 bp of 5' untranslated region, and 171 bp of 3' untranslated region followed by a putative poly(A) tail. The coding region of the cDNA was inserted into an expression vector that was used to obtain the recombinant protein from Escherichia coli cells. The recombinant enzyme (44 kDa) had a specific activity and other properties similar to those of native bovine brain inositol polyphosphate 1-phosphatase. It hydrolyzed both inositol phosphate substrates and was inhibited by lithium ions. The enzyme shows minimal sequence similarity to inositol monophosphate phosphatase, the other enzyme inhibited by lithium ions in the signaling pathway.

Preparative‐enzymatic formation of cytidine 5′‐monophosphosialate by integrated cytidine 5′‐triphosphate regeneration

AbstractThe preparative formation of cytidine 5′‐triphosphate by an enzymatic process with adenylate kinase and pyruvate kinase is described. The enzymes may either be used free, in the “MEEC” technique, or immobilized on a synthetic copolymer of vinyl acetate and N,N′‐divinylethyleneurea (“VA‐Epoxy”). Cytidine 5′‐monophosphosialate synthase (preferably isolated from calf brain) and inorganic pyrophosphatase were used to prepare the activated neuraminic acid cytidine 5′‐monophosphosialate (CMP‐Neu5Ac) from CTP and neuraminic acid in good yield. Finally, both processes could be integrated and led to an in situ approach which, owing to moderate yields, requires further development.

Anti-imipramine antibodies recognize endogenous serotonin uptake and imipramine binding inhibitors

Calf brain and human platelet extracts purified by Bio-Gel P 2 column chromatography contained substances that inhibited serotonin uptake and 3H-imipramine binding. Some of these endogenous substances were also recognized by rabbit antibodies produced against imipramine. The data suggest the possible existence of endogenous serotonin uptake modulators, which may possess a partial molecular structure similar to that identified by the antibodies.

Partition of synaptic membranes in aqueous two-phase systems at subzero temperatures by using anti-freeze solvent

The freezing point of aqueous two-phase (liquid-liquid) systems containing water, dextran and poly(ethylene glycol) has been lowered by including glycerol. Biological membranes, obtained by fragmentation of a crude synaptosomal preparation from calf brain cortex, have been included in the two-phase systems. The effects of temperature and the concentration of glycerol on the partition of the membranes within the systems have been investigated. Considerable stabilisation of the membranes was noticed when they were partitioned at −10°C compared with 0°C. The influences of glycerol, ethylene glycol, N, N-dimethylformamide and tetrahydrofuran on the phase-forming properties of the systems and on enzyme activities are also presented. Possible use of the above systems for studies and separation of biological membranes are discussed.

Effects of Dietary Corn Oil and Fish Oil Concentrate on Lipid Composition of Calf Tissues

Lipid composition of calf blood plasma, liver platelets, muscle, heart, and brain was measured, as affected by high dietary intake of linoleic acid from corn oil or of polyunsaturated fatty acids from fish oil concentrate. Plasma total lipids, phosphatidylcholine, and cholesteryl esters were reduced by corn oil and fish oil concentrate. Dietary fatty acid composition had no influence on percentage distribution of the major phospholipid components of liver, heart, muscle, and brain, but did alter the polyunsaturated fatty acid composition of major phospholipids in plasma, liver, platelets, muscle, and heart. In general, high linoleic acid intake increased linoleic acid and decreased oleic, arachidonic, and linolenic acids in tissue phospholipids, and fish oil concentrate high in eicosapentaenoic and docosahexaenoic acids increased phoslipid concentrations of these fatty acids. The fatty acid composition of brain phosphatidylcholine and phosphatidyl-ethanolamine was relatively resistant to dietary lipid alterations. The fatty acid changes in tissue phospholipids that resulted from dietary lipid alterations may have important implications in eicosanoid metabolism and in the structure and function of cell membranes.

Interaction of substance P, compound 4880 and mastoparan with the α-subunit C-terminus of G protein

Pretreatment of purified calf brain G proteins with activated pertussis toxin or antibodies raised against the C-terminus of their alpha subunits prevented the increase in GTPase activity induced by substance P, compound 48/80 and mastoparan. These results suggest that these mast cell secretagogues activate G proteins directly via an interaction with the C-terminus of alpha subunits of G proteins by mimicking the agonist-liganded receptors.

Chemical modification of adenosine A 1 receptors : Implications for the interaction with R-PIA, DPCPX and amiloride

Amiloride, a potassium sparing diuretic, inhibits the specific binding of [ 3H]8-cyclopentyl-1,3-dipropylxanthine (DPCPX) and [ 3 H]N 6- R-1- phenyl-2- propyladenosine (PIA) to adenosine A 1 receptors in calf brain. This interaction is different from the agonist-receptor or the antagonist-receptor interaction as Na + and H + counteract the inhibitory effect of amiloride whereas these ions hardly affect the binding of the classic A I receptor ligands. In the present study, the effects of protein modifiers on the equilibrium inhibition constant of amiloride are compared with effects of these reagents on the affinities of DPCPX and PIA. It is demonstrated that the affinities of amiloride and [ 3H]DPCPX are changed after treatment with a carboxyl-modifying reagent but unaffected by modification of histidyl, arginyl and cystein residues. The maximal binding capacity of [ 3H]DPCPX is enhanced by sulfhydryl modification, whereas the number of [ 3H]DPCPX binding sites is reduced by treatment with a histidine-modifying reagent. The histidyl residues of the [ 3H]DPCPX binding site can be partially protected against modification by 300 μM amiloride, present during treatment of the membranes. An equivalent concentration of 8-phenyltheophylline results in complete protection. The apparent affinity of PIA is altered by modification of histidyl, carboxyl, arginyl and cystein residues. In the latter two cases, uncoupling of the G protein seems to be the major reason for the decrease in affinity of PIA. The results suggest that amiloride is an A 1 antagonist with binding characteristics that differ from the classic A 1 antagonists such as DPCPX.

Interaction of amiloride and its analogues with adenosine A 1 receptors in calf brain

Amiloride, a potassium sparing diuretic, is known to interact with a number of ion transport systems, receptors and enzymes. Here, we report on the interaction between this drug and the adenosine A 1 receptor as present in calf brain membranes. Adenosine A 1 receptors are characterized by a subnanomolar affinity for the antagonist [ 3H]8-cyclopentyl-1,3-dipropylxanthine ([ 3H]DPCPX) and the agonist [ 3 H]N 6- R-1- phenyl-2- propyladenosine ([ 3H]PIA). Amiloride displaces both agonist and antagonist binding with a K i value in the low micromolar range. This inhibition is counteracted by NaCl and protons, in contrast to the binding of [ 3H]PIA and [ 3H]DPCPX. The results suggest that amiloride interacts with the adenosine A 1 receptor at a site distinct from the ligand binding site. In order to elucidate the role of one of the ion transport systems known to be inhibited by amiloride, eight amiloride analogues with different sensitivities for these systems were tested. The potency and order of potency of these compounds towards adenosine A 1 receptors excludes the involvement of the epithelial Na + channel, Na + H + exchanger or Na + Ca 2+ exchanger.

Evidence for the presence of water within the hydrophobic core of membranes

The photoreactive ganglioside derivative N-diazirinyl-lyso-G MI was incorporated into liposomes and calf brain microsomes. After photoactivation at 350 nm it was found to dimerize with phospholipids such as phosphatidylcholine and phosphatidylserine and with cholesterol. The predominant covalent reaction product, however, was the alcohol, resulting from the reaction with water. It amounted to about 45% of the covalent reaction products in calf brain microsomes and to about 58% in pure phosphatidylcholine liposomes. Based on the temperature dependence of the photoreaction of N-diazirinyl-lyso-G MI in liposomes consisting of 1,2-dipalmitoyl- sn-glycero-3-phosphoryl-choline or 1,2-distearoyl- sn-glycero-3-phosphorylcholine and on affinity labeling experiments with cholera toxin we propose that the predominant reaction of N-diazirinyl-lyso-G MI with water is due to the presence of water within the hydrophobic core of artificial and biological membranes.

Tubulin assembly probed with antibodies to synthetic peptides

Antibodies to synthetic peptides from the alpha and beta-tubulin sequences were employed to study zones of this protein active in microtubule assembly. In purified calf brain tubulin, six short sequences, selected according to their hydrophilicity and conservation, were found to be accessible to their affinity-purified immunoglobulin G (IgG) antibodies, in a competition radioimmunoassay performed under non-assembly native conditions. This indicated that the six sequences are exposed on the surface of the tubulin alpha beta heterodimer. IgG antibodies to the alpha(430-443) and beta(412-431) sequences perturbed substoichiometrically the assembly of purified tubulin, inducing microtubule bundling and the formation of opened up structures. These positions, which are close to the C termini, were accessible to the anti-peptide antibodies in taxol-induced microtubules, Zn2(+)-induced tubulin sheets, Mg2(+)-induced tubulin rings and in PtK2 cell microtubules. This, together with the comparison of the sizes and gross shapes of the antibody probes and microtubules, suggested that these sequences might be located at the protruding parts of the protofilaments. Antibodies to positions alpha(155-168) did not react with microtubules, while the equivalent zone beta(153-165) was accessible. The alpha(214-226) and beta(241-256) sequences were antigenically occluded in the taxol microtubules, Zn2(+)-induced sheets and Mg2(+)-induced ring arrays, as well as in native microtubules from PtK2 cells, though they became reactive by fixation. This result strongly suggested that these two zones are close to tubulin-tubulin contact sites. A working model is proposed in which the positions alpha(214-226) and beta(241-256) are close to the axial contacts between heterodimers, which lead to protofilament formation, while the positions alpha(241-256) and beta(214-226) are suggested to be related to the alpha-beta binding interface within the heterodimer.

Direct Thin-Layer Chromatographic Method for Isolation of Gangliosides

Abstract A simple procedure is developed for obtaining a ganglioside mixture. The scheme of the method is as follows: the sample was homogenized and extracted twice with cyclohexane (first extraction). The residue was reextracted with a mixture of chloroform /methanol = 1:3 (v/v) (second extraction). The extracts were collected after solvent evaporation. The residue was dissolved and applied onto a thin layer of silica gel according to the requirements of the preparative thin-layer chromatography. After the zone separation the visualized spots were scrapped and transferred to a column and eluted with a mixture of chloroform/methanol with increasing polarity. The eluate was concentrated and controlled for purity and composition by HPTLC. This procedure has been successfully applied to fractionation of ganglioside mixtures from human, calf and rat brains.

Role of the phospholipid environment in modulating the activity of the rat brain synaptic plasma membrane calcium ATPase

The role of the phospholipid environment in modulating the activity of the rat brain synaptic plasma membrane (SPM) Ca2(+)-ATPase was investigated by its reconstitution into different phospholipids. Retention of activity of the solubilized Ca2(+)-ATPase depended on addition of exogenous phospholipids. As the cholate concentration used for solubilization of native SPM increased, a larger excess of exogeneous phospholipids, relative to membrane protein, had to be added to maintain optimal activity. Highest ATP-dependent Ca2+ transport activity was obtained when reconstitution was carried out in calf brain phospholipids (BPLs) followed by soybean phospholipids (SPLs) and the lowest in egg PC; reconstitution at a 40:1 weight ratio of exogenous phospholipids to native SPM protein resulted in ATP-dependent Ca2+ transport of 40.0 +/- 4.16, 23.4 +/- 8.48, and 11.54 +/- 2.31 nmol of Ca2+ (mg of protein)-1 (5 min)-1, respectively. Partial substitution of egg PC with BPLs led to an increase in the activity of the reconstituted Ca2+ pump. The highest ATP-dependent Ca2+ uptake was obtained when ratios of 15:25 or 10:30 egg PC to BPLs were used. Testing the individual phospholipids participating in the BPL mixture showed that addition of PS to egg PC led to a consistent increase in Ca2+ pump activity. Substitution of 50% of the PC with PS resulted in a 3.8-fold higher ATP-dependent Ca2+ uptake than that obtained in egg PC alone. No other phospholipid tested--PE, SM, or PI--had a similar effect. Increasing the proportion of PS within the BPL mixture above its original content led to a gradual decrease in the reconstituted SPM Ca2+ pump activity. Enrichment of asolectin with PS led first to increased Ca2+ pump activity; then, as the proportion of PS increased, Ca2+ transport of the reconstituted pump decreased. An increased proportion of PE, SM, or PI within the BPLs or asolectin, above their original contents, resulted in decreased Ca2+ transport. These results indicate that optimal SPM Ca2+ pump activity requires the combined presence of a critical amount of PC and PS within the reconstituted membrane.

Interactions of tubulin with guanylyl-(beta-gamma-methylene)diphosphonate. Formation and assembly of a stoichiometric complex.

Complete replacement of the nucleotide on the exchangeable binding site of purified calf brain tubulin by the non-hydrolyzable GTP-analogue guanylyl-(beta,gamma-methylene)diphosphonate (GMPPCP) has been achieved by treatment of tubulin-GDP with phosphodiesterase-free alkaline phosphatase. GMPPCP binds to tubulin with a low affinity relative to GTP or GDP. Binding of the analogue is linked to magnesium ion concentration and, like the binding of other guanine nucleotides, is promoted by high concentrations of glycerol. The complex of pure tubulin and GMPPCP readily assembles at 37 degrees C into microtubules or curled ribbons of protofilaments, depending on buffer composition. Assemblies are cold-reversible at 0-2 degrees C, and multiple reversible assemblies can be observed during repeated heating/cooling cycles.

Capacitive function of the spleen during electrical stimulation of the ventral medulla oblongata

New dialkylaminoacyl phenothiazine derivatives (DAC) were compared with their dialkylaminoalkyl analogues (neuroleptics chlorpromazine, trifluoperazine and fluphenazine) as well as with anti-arrhythmia drugs ethmozine and ethacizine for their receptor-blocking potencies. It was established that DAC are significantly less potent with dopamine alpha 1-adrenergic and H1-histamine receptors of calf and rabbit brain, which can explain the absence of neuroleptic effect of DAC drugs. DAC affinities to muscarinic and alpha-adrenergic receptors of both types are very similar to those of ethmozine and ethacizine. New DAC substance G-512 (chlorpromazine analogue) demonstrated high affinity to M1-muscarinic receptors of rabbit brain cortex (Ki = 4.2 nM) and to M2-muscarinic receptors of the rabbit heart (Ki = 48 nM).