Preparation of enriched fractions from cerebral cortex containing isolated, metabolically active neuronal and glial cells

1. A procedure has been developed for the separation of intact metabolically active neuronal and glial cells in bulk from rat cerebral cortex. Separation depended on dispersion of the tissue in a Ficoll medium followed by centrifugation on a discontinuous Ficoll gradient. Up to 1.5x10(7) neuronal cells could be collected from 12 brains within 3hr. The morphological appearance of these cells seemed good, and the fraction was 8.5-fold purified in terms of dry weight. Average dry weight per neuron was 2300mumug. Maximum glial contamination of the neuronal fraction was 11% as determined by carbonic anhydrase measurements. The glial fraction was free from neurons but contained various subcellular contaminants. 2. Concentrations of nucleic acids, phospholipid, protein and phosphoprotein were determined in the separated fractions. The neuronal fraction was richer than the glial in all except phospholipid. Succinate dehydrogenase was equally distributed between neurons and glia but the neuronal fraction was 1.8-fold enriched in cytochrome oxidase. 3. Measurement of respiration by the cells showed an endogenous uptake of 117mmumoles of oxygen/mg./hr. in neurons, and 173mmumoles of oxygen/mg./hr. in glia. Addition of substrate at 10mm stimulated uptake to similar values in both fractions. With glucose it was 390, with pyruvate 355, and with glutamate 215mmumoles of oxygen/mg./hr. This represented a larger stimulation of neuronal than of glial respiration compared with the basal level. 4. Respiration in cell suspensions was 70-80% of that of slices, whereas fractionated tissue homogenates had respiratory rates of only one-third those of the cell suspensions. Lactate dehydrogenase content of cell suspensions was maintained during gradient centrifugation and washing. 5. The possible uses of isolated cell preparations are discussed.     

Phosphatidylinositol synthetase activities in neuronal nuclei and microsomal fractions isolated from immature rabbit cerebral cortex

The synthesis of phosphatidylinositol was studied using a nuclear fraction N1, a microsomal fraction P3, rough (R) and smooth (S) microsomal fractions and a microsomal fraction P derived from isolated nerve cell bodies. Each fraction was prepared using cerebral cortices of 15-day-old rabbits. In assays using CDP-diacylglycerol (prepared from egg phosphatidylcholine) and myo[3H]inositol at pH 7.4, fraction N1 had the highest maximal specific rates of phosphatidylinositol synthetase (EC 2.7.8.11) (expressed per mumol phospholipid in the fraction). However the three microsomal fractions achieved maximal specific activities at liponucleotide concentrations close to 50 microM, while fraction N1 required 200 microM concentrations. In certain cases (25-120 microM CDP-diacylglycerol, and at higher pH values) fraction R had specific activities which equalled or surpassed those of N1. However, with respect to inositol, fraction N1 had a distinctly lower Km than was shown for fractions R or P3. Each of the microsomal fractions and N1 required Mg2+ for the reaction, but for N1, maximal rates could be sustained at 0.1 mM, while for the microsomal fractions the optimal Mg2+ concentration was 1 mM. For each fraction Mn2+ could not replace Mg2+ in the reaction and Mn2+ was inhibitory. The optimal pH for the reaction was between 8.0 and 9.0. Phosphatidylinositol synthetase could also be shown using fraction N1 enriched in endogenous CDP-diacylglycerol. The relatively high specific activities of fraction N1, and the differences found between N1 and the microsomal fractions, for optimal CDP-diacylglycerol and Mg2+ concentrations and for Km values for inositol, support the existence of a neuronal nuclear phosphatidylinositol synthetase.     

Oxidative events in neuronal and glial cell-enriched fractions of rat cerebral cortex

The aim of this work was to investigate how neurons and glial cells separated from rat brain cortex respond to “in vitro” oxidative stress induced by incubation of the cellular fractions in the presence of prooxidant mixtures; in addition, the endogenous enzymatic antioxidant capacity of the purified fractions was investigated. Neuronal and glial cell-enriched fractions were obtained from rat cerebral cortex following passages of the tissue through meshes and centrifugations. The following parameters were evaluated: antioxidant enzymes superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSHPx), and glucose-6-phosphate dehydrogenase (G6PDH); lipid peroxidation products (TBARS) prior to (basal) and after (iron-stimulated) incubation with a mixture of iron and ascorbic acid; intracellular production of reactive oxygen species (ROS) using a fluorescent probe, dichlorofluorescin-diacetate, in basal, iron-stimulated, and menadione stimulated conditions. SOD and GSHPx activities showed no significant changes between neurons and glia, whereas CAT and G6PDH activities were found to be significantly lower in glia than in neurons. TBARS levels were significantly lower in the glial fraction than in neurons, both in basal and iron-stimulated conditions. ROS production showed no differences between neurons and glia in both basal and menadione-stimulated conditions. Iron-stimulation produced a marked increase in ROS production, limited to the neuronal fraction, with the glial values being similar to the basal ones. Our conclusion is that glia and neurons isolated from rat cerebral cortex show a similar pattern of the most important antioxidant enzymes and of their basal ROS production, whereas glia is more resistant in “oxidative stress” conditions.     

Activity and subcellular distribution of phospholipase A1 from neuronal cell-enriched fractions of the rabbit cerebral cortex

Glycerophosphatides, specifically labeled either in the 1 or in the 2 position, were used to measure the activity of neuronal phospholipase A₁ and to investigate the subcellular distribution of the enzyme. The microsomes were found to possess the highest phospholipase activity, with a threefold increase as compared to the cell homogenate. A considerable enzymatic activity could still be observed in the plasma membranes isolated from the neuronal-enriched cell fraction. Microsomal phospholipase possessed the highest activity with phosphatidylcholine, whereas phosphatidylserine was cleaved at a much lower rate. The rate of release of labeled fatty acids from the substrates by the microsomal phospholipase decreased with increasing degree of unsaturation of the fatty acids at the 1 position. The presence of plasmalogens and of alkylacyl analogues in the incubation mixture caused an appreciable inhibition of the hydrolysis of the diacyl glycerophosphatides.     

Phospholipid composition and fatty acid patterns of isolated phospholipids from rabbit reticulocyte mitochondria

The phospholipid composition and fatty acid patterns of individual phospholipid classes were determined in mitochondria from rabbit reticulocytes. Compared to mitochondria from rat liver reticulocyte, mitochondria exhibit about twice the amount of phospholipids. The phospholipid pattern of reticulocyte mitochondria (phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol and cardiolipin) is comparable with other mitochondrial species. Mitochondrial fractions from reticulocytes are characterized, however, by an additional content of sphingomyelin. This sphingomyelin differs in its fatty acid composition from the sphingomyelin of the plasma membrane. The fatty acid patterns of all other phospholipids essentially correspond to those of mitochondria from other sources and to those of plasma membranes as well.     

The fatty acid composition of fern lipids

Fatty acid compositions of the lipids isolated from the pinnae of four fern species show that they differ from those of the lipids in leaves of higher plants in having C-20 polyunsaturated acids, mainly arachidonic acid. As in higher plants, the ω-3 polyunsaturated acids are concentrated in the monogalactosyl diglycerides. Variations are found both in the fatty acid compositions and in the monogalactosyl/digalactosyl diglyceride ratio during the growing season.     

The fatty acid composition of leech lipids

• 1.1. The constituent fatty acids of the neutral and phospholipids of Macrobdella ditetra, Nephelopsis obscura, Philobdella gracilis and Hirudo medicinalis have been determined.
• 2.2. Unsaturated fatty acids predominated in both neutral and phospholipid fractions of all leech species examined.
• 3.3. Arachidonic acid (20:4) was the most prevalent fatty acid in all species, accounting for as much as 36.7% of the total phospholipid fatty acids.

     

The formation of phosphatidic acid de novo: a comparison of activities in neuronal nuclei and microsomes isolated from immature rabbit cerebral cortex

The formation of phosphatidic acid from sn-glycerol 3-phosphate was studied in neuronal nuclear fraction N1 and a microsomal fraction P3, isolated from cerebral cortices of 15-day-old rabbits. Two assays were used, employing dithiothreitol, MgCl2, NaF and (A) sn-glycerol 3-phosphate, [14C]oleate, ATP and CoA or (B) sn-[3H]glycerol 3-phosphate and oleoyl-CoA. In both assays fraction N1 had specific rates of phosphatidic acid labelling (expressed per mumol phospholipid in the fraction) which were 5- to 6-times the corresponding values for P3. In contrast to N1, the formation of phosphatidic acid by fraction P3 was more sensitive to inhibition at high concentrations of oleoyl-CoA and was greatly dependent upon the presence of NaF. In the absence of this salt, P3 showed decreased phosphatidate formation and increased levels of radioactive monoacylglycerols. Using cerebral cortex, rough (R) and smooth (S) microsomal fractions were prepared, as was a microsomal fraction P from isolated nerve cell bodies. P had specific rates of phosphatidic acid labelling which were 2-3 times the values for P3, but were about 50% of the N1 values. This indicates a concentration of phosphatidate synthesis in the nucleus within the nerve cell. Specific rates for fraction R were higher and were similar to those of N1. In S, P3 and R the specific rates of phosphatidic acid synthesis paralleled specific RNA contents and indicated a location for phosphatidic acid synthesis within the rough endoplasmic reticulum.     

The acylation of 1-acyl-sn-glycero-3-phosphate by neuronal nuclei and microsomal fractions of immature rabbit cerebral cortex

The acylation of 1-acyl-sn-glycero-3-phosphate to form phosphatidic acid was studied using a neuronal nuclear fraction N1 and microsomal fractions P3, R (rough), S (smooth), and P (neuronal microsomes from nerve cell bodies) isolated from cerebral cortices of 15-day-old rabbits. The assays contained this lysophospholipid, ATP, CoA, MgCl2, NaF, dithiothreitol, and radioactive palmitate, oleate, or arachidonate. Of the subfractions, N1 and R had the highest specific activities (expressed per micromole phospholipid in the fraction). The rates with oleate were two to four times the values seen for phosphatidic acid formation from sn-[3H]glycero-3-phosphate and oleoyl-CoA. Using oleate or palmitate, fraction R had superior specific rates to N1 at low lysophosphatidic acid concentrations. With increasing lysophospholipid concentrations the specific rates of N1 and R came closer together and maintained at least a twofold superiority over fraction P. Fraction S had the lowest specific rates of phosphatidic acid formation. Fractions N1, R, and P showed a preference for palmitate and oleate over arachidonate, particularly at low concentrations of lysophosphatidic acid. For N1 and R, the preference was also more marked at higher concentrations of fatty acid. Thus a selectivity for saturated and monounsaturated fatty acids was shown in the formation of phosphatidic acid, as was a concentration of acylating activity in the neuronal nucleus and the rough endoplasmic reticulum.     

Electron microscopy of two subcellular fractions isolated from cerebral cortex homogenate

The sporulation process in Bacillus subtilis has been studied principally with KMnO(4) fixation, but also, for the purpose of comparison, with OsO(4) and mixtures of both fixatives. At a very early stage, the pre-spore is seen to consist of what seems to be the nuclear material and granular substance, surrounded by a layer of dense material destined to become the innermost layer of the spore coat. At a subsequent stage, a light interspace is observed that is destined to become the spore cortex. The mature spore shows a very complex structure. The spore coat is composed of three layers, the middle layer of which consisted of 5 to 8 lamellae of thin membranes and interspaces, both about 20 to 25 A thick. Between the inner layer of the spore coat and the spore cortex, a thin membrane with an affinity to the cortex can be observed. The spore coat is enclosed within two envelopes, one loosely surrounding the core, and the other adhering to it. The process of spore maturation has been studied in detail. Certain peculiar cellular structures have been observed that seemed to represent features of abnormal sporulation processes.     

Polar lipids and fatty acid composition ofThermus strains from New Zealand

The polar lipids and fatty acid composition ofThermus aquaticus YT-1 and YS 041,T. filiformis Wai33 A1 and eighteen isolates from New Zealand, several of which are attributed toT. filiformis, were compared to complement the taxonomy of these organisms. The polar lipid patterns were essentially similar in all strains and consisted of one major phospholipid and one major glycolipid. The fatty acid analysis produced three basic groups corresponding toT. filiformis Wai33 A1,T. aquaticus and the third to the other New Zealand strains. The presence of hydroxy fatty acids is reported inThermus spp. for the first time.     

The fatty acid composition of Ulothrix aequalis lipids

Fatty acid composition of the total lipids isolated from the fresh-water green alga Ulothrix aequalis shows that they resemble marine green algal lipids in having a high proportion of 16:4 ω-3 but differ in having only trace amounts of 18:4 ω-3. The distribution of ω-3 acids in the MGDG and DGDG fractions resembles that in green seaweeds and higher plants with the 16:4 ω-3 distribution in the Ulothrix fractions resemblmg that of 18:4 ω-3 in the corresponding fractions of the seaweeds.