Showing papers in "Neurochemical Research in 1981"
TL;DR: Evidence is presented in support of the working hypothesis that “prenervous” neurotransmitters directly participate in cell-cell interactions occurring during the first several cleavage divisions of sea urchin embryos, a function which may occur during the early development of higher animals as well.
Abstract: Evidence is presented in support of the working hypothesis that "prenervous" neurotransmitters directly participate in cell-cell interactions occurring during the first several cleavage divisions of sea urchin embryos, a function which may occur during the early development of higher animals as well. This intercellular signaling could be a link in the evolutionary progression from the use of these substances as intracellular regulators to their participation in cell-cell interactions occurring during synaptic transmission.
95 citations
TL;DR: All the data indicate that the prior injection of CDP-choline stimulates the choline phosphotransferase reaction of brain towards synthesis of phosphatidylcholine and prevents the release of free fatty acids, particularly of arachidonic acid, associated with ischemia.
Abstract: Brain ischemia was produced in gerbils (Meriones unguiculatus) by the bilateral ligation of the carotid arteries. Definite changes in the energy status of brain demonstrated that carotid occlusion was effective. Five minutes before ligation, an intraventricular injection of either saline or cytidine diphosphate choline (CDP-choline, 0.6 μmol/brain, 3μl) was given to groups of animals. Control animals, with and without CDP-choline, together with the ischemic groups, were decapitated directly into liquid nitrogen; 10 min after arterial ligation. Brain free fatty acids, neutral lipids and phospholipids, which were labeled in vivo by the intraventricular injection of [1-14C] arachidonic acid (0.4–0.6 μCi, 6–9 nmol) 2 hr prior to ligation, were extracted, purified, and separated by thin-layer chromatographic procedures. The CDP-choline treatment noticeably corrected the increase of total and individual fatty acids due to ischemia and the increase of their radioactivity content. The changes in neutral lipids, particularly in the diacyl glycerol fraction, were also corrected by the injection of the nucleotide. CDP-choline partially reversed the decrease of brain phosphatidylcholine and of its labeling, which was due to ischemia. All the data indicate that the prior injection of CDP-choline stimulates the choline phosphotransferase reaction of brain towards synthesis of phosphatidylcholine and prevents the release of free fatty acids, particularly of arachidonic acid, associated with ischemia.
92 citations
TL;DR: Low levels of antibodies reacting with MAG were detected in three rabbits with experimental allergic encephalomyelitis induced by injection of purified myelin in complete Freund's adjuvant, indicating that the antigenic site(s) is not accessible in the intact membranes, but can be exposed by treatment with detergent or partial purification.
Abstract: Rabbits were immunized with the myelin-associated glycoprotein (MAG) that had been purified from isolated rat brain myelin by selective extraction with lithium diiodosacicylate (LIS) and phenol followed by preparative SDS gel electrophoresis. Antibodies to MAG were detected qualitatively by immunodiffusion and quantitatively by a double antibody assay utilizing [3H]fucose-labeled MAG as antigen. The antisera were capable of precipitating between 300 and 500 μg of MAG/ml of serum under the conditions of the assay. Preincubation of the anti-MAG serum with other glycoproteins or glycolipids did not inhibit the precipitation of labeled MAG. Similarly, preincubation of the antiserum with LIS-phenol extracts of non-neural tissues did not inhibit the immune precipitation of MAG. The specificity of the antiserum was also indicated by the selective double antibody precipitation of MAG from solubilized whole myelin that contained a heterogeneous mixture of [3H]fucose-labeled glycoproteins. The antibodies to MAG were not effectively absorbed by whole brain homogenate or purified myelin, indicating that the antigenic site(s) is not accessible in the intact membranes, but can be exposed by treatment with detergent or partial purification. Low levels of antibodies reacting with MAG were detected in three rabbits with experimental allergic encephalomyelitis induced by injection of purified myelin in complete Freund's adjuvant.
74 citations
TL;DR: The findings indicate the widespread occurrence of neurofilament proteolysis following calcium influxes into CNS and PNS tissues, and the parallel breakdown of glial filaments and loss of GFA protein subunits suggest the presence of additional calcium-activated proteases(s) in astroglial cells.
Abstract: Disruptive effects of calcium upon neurofilaments and glial filaments were studied in white matter of rat optic nerve and spinal cord and in rat peripheral nerve. Filament ultrastructure and tissue protein composition were compared following a calcium influx into excised tissues. A calcium influx was induced by freeze-thawing tissues in media containing calcium (5 mM) while control tissues were freeze-thawed in the presence of EGTA (5 mM). Experimental and control tissues were either fixed by immersion in glutaraldehyde and processed for electron microscopic examination or homogenized in a solubilizing buffer and analyzed for protein content by SDS-polyacrylamide gel electrophoresis. Morphological studies showed that calcium influxes led to the loss of neurofilaments and glial filaments and to their replacement by an amorphous granular material. These morphological changes were accompanied by the loss of neurofilament triplet proteins and glial fibrillary acidic (GFA) protein from whole-tissue homogenates. In addition, a calcium-sensitive 58,000-mol-wt protein was identified in rat optic and peripheral nerve. The findings indicate the widespread occurrence of neurofilament proteolysis following calcium influxes into CNS and PNS tissues. The parallel breakdown of glial filaments and loss of GFA protein subunits suggest the presence of additional calcium-activated proteases(s) in astroglial cells.
68 citations
TL;DR: Inhibition of SOD activity by ethanol may allow an accumulation of cytotoxic O2− radicals; this may account for some nervous system disorders during alcohol intoxication.
Abstract: The effect of acute and chronic ethanol administration on rat brain superoxide dismutase (SOD) activity was studied. Intraperitoneal injections of ethanol led to an inhibition of SOD activity. When ethanol was fed as the sole fluid, the SOD activity decreased progressively, reaching a plateau after 6 weeks of treatment. Withdrawal of ethanol produced a recovery of control values within 48 hr. SOD activity was also decreased in rats born from ethanol-drinking mothers. Inhibition of SOD activity by ethanol may allow an accumulation of cytotoxic O2
− radicals; this may account for some nervous system disorders during alcohol intoxication.
59 citations
TL;DR: The findings show that even slowly penetrating amino acid levels can be increased in brain after parenteral administration of large doses.
Abstract: Taurine, aspartic acid, glutamic acid, glycine, and GABA were administered either intragastrically or in liquid diets to mice and rats. This resulted in a great increase in the plasma concentration of the administered amino acid, with plasma levels remaining elevated for several days.
58 citations
TL;DR: It was found that repeated haloperidol treatment reduced GAD holoenzyme activity in the substantia nigra pars reticulata and the cofactor saturation was found to be uneven in the discrete nuclei.
Abstract: Glutamate decarboxylase (GAD) activities with and without added pyridoxal-5′-phosphate were determined in discrete brain nuclei of freeze-dried samples. The distribution of GAD holoenzyme activity as well as the cofactor saturation, was found to be uneven in the discrete nuclei. In addition, it was found that repeated haloperidol treatment reduced GAD holoenzyme activity in the substantia nigra pars reticulata.
52 citations
TL;DR: An extraction procedure for the isolation of proteins from the extracellular fluid (ECF) of goldfish brain was developed and applied in an investigation of the time course and pattern of labeling of ECF proteins, suggesting that the gold fish brain intracellular and extacellular fluids contain relatively comparable levels of proteins.
Abstract: An extraction procedure for the isolation of proteins from the extracellular fluid (ECF) of goldfish brain was developed and applied in an investigation of the time course and pattern of labeling of ECF proteins. The results indicate that two out of the many protein bands present, which migrated at 32,000 and 26,000 daltons on SDS-polyacrylamide electrophoretic gels, could incorporate as much as 50% of the label of the ECF fraction, even though their concentration was only 14%. Measurements of the protein content of the ECF and its volume (24% of the brain) by the inulin method were used to calculate the protein concentration in the extracellular space of goldfish brain. This gave a value of 1.6–2%, i.e., about 50% of the value obtained for the protein concentration of the cytoplasmic fraction devoid of particulate matter. Such a result suggests that the goldfish brain intracellular and extracellular fluids, separated by the neural membranes, contain relatively comparable levels of proteins.
45 citations
TL;DR: In this paper, the authors compared taurine and GABA uptakes in brain slices under identical experimental conditions, and found that the uptakes of these three structurally related amino acids were all saturable, consisting of one low-and one high-affinity transport component.
Abstract: Hypotaurine uptake was compared to taurine and GABA uptakes in brain slices under identical experimental conditions. The slices effectively concentrated both hypotaurine and GABA from the medium, whereas taurine was taken up more slowly. The uptakes of these three structurally related amino acids were all saturable, consisting of one low-and one high-affinity transport component. The kinetic parameters of hypotaurine uptake were of the same order of magnitude as those of GABA uptake. All uptake systems were sensitive to temperature, metabolic poisons, and sodium omission. Hypotaurine uptake was inhibited by GABA,l-2,4-diaminobutyric acid (l-DABA), cysteic acid, and β-alanine, but not by taurine. Taurine uptake was strongly reduced by hypotaurine, β-alanine, andl-DABA, as well as by GABA, whereas GABA uptake was affected only by cystamine,l-DABA, and nipecotic acid. The uptake processes of hypotaurine, taurine, and GABA were thus fairly similar and showed properties characteristic for neurotransmitter uptake. Hypotaurine uptake resembled more GABA than taurine uptake. The present inhibition studies suggest that there may exist only one common two-component transport system for these three amino acids.
38 citations
TL;DR: Kinetic analysis of the binding process indicated a single high-affinity system with a capacity of approximately 20 pmoles/mg protein in all the membrane fractions.
Abstract: Binding ofl-[3H]glutamate to membranes from whole chick retina and from subcellular fractions enriched with photoreceptor terminals (P1), or terminals from the inner plexiform layer (P2) was studied. Na+-dependent and Na+-independent binding to these membranes was demonstrated. Na+-independent binding was stereospecific. Kinetic analysis of the binding process indicated a single high-affinity system (KB=0.55 μM) with a capacity of approximately 20 pmoles/mg protein in all the membrane fractions. [3H]Glutamate binding to P1 and P2 fractions was effectively displaced by several structural analogues of glutamate. Glutamate diethyl-ester appreciably displaced binding, whereas kainic acid did not displace bound glutamate. Data indicate the binding of [3H]glutamate to physiologically relevant receptors in the chick retina.
34 citations
TL;DR: The four cholinergic parameters are statistically correlated throughout all the areas of the medulla which were studied, and are found to be highest in the hypoglossal nucleus and the dorsal motor nucleus of the vagus.
Abstract: Quantitative measurements were made of choline acetyltransferase (CAT) activity, acetylcholinesterase (AChE) acitivity and cholinergic muscarinic receptor binding ([3H]QNB) in eight areas of a cross-section of the rat medulla oblongata. A fourth cholinergic parameter, high-affinity choline uptake, was measured in three groups of these areas. CAT, AChE and [3H]QNB binding were found to be highest in the hypoglossal nucleus and the dorsal motor nucleus of the vagus; the lowest value was in the area which contains the inferior olive and the corticospinal tract. The distribution of AChE and CAT acitivities varied approximately 7- to 10-fold among the eight regions examined, whereas that of the muscarinic receptor varied only about 4-fold. The Na+-dependent high-affinity choline uptake varied approximately 20-fold from the region with the lowest activity (inferior olivary nucleus and corticospinal tract) to that with the highest activity (tissue areas containing the dorsal motor nucleus, hypoglossal nucleus, nucleus of the solitary tract and nucleus cuneatus). The four cholinergic parameters are statistically correlated throughout all the areas of the medulla which were studied.
TL;DR: The patterns of brain enzymes linked to energy metabolism have been determined in rats aged between 3 and 21 months and compared to those of the developing brain as an estimate of the senescent energy capacity of this organ.
Abstract: The patterns of brain enzymes linked to energy metabolism have been determined in rats aged between 3 and 21 months and compared to those of the developing brain as an estimate of the senescent energy capacity of this organ. During aging, pyruvate kinase increases, pointing towards an enhancement of the glucose-dependence of this organ. However, NAD-isocitrate dehydrogenase declines, suggesting a reduction of Krebs cycle activity in the aged rat brain. An increase in cytoplasmic NAD-malate dehydrogenase found during aging could provide an alternative mechanism of NAD recovery.
TL;DR: Protein malnutrition markedly affected the composition of acyl-linked fatty acids in the synaptic membranes and the increases in the ratio of 22∶6 (n-3)/22∶5 ( n-6) fatty acids were especially compromised.
Abstract: The acyl-linked fatty acid composition of the major phospholipid species in rat cortical synaptic membranes was determined at various stages of development. For most species there was a decrease during development in the short chain saturated fatty acids, 14:0 and 16:0, an increase in 18:0 and 22:6 (n-3) and an increase in ratio of 22:6 (n-3)/22:5 (n-6). Pups were protein deprived by feeding the dams a 12% casein diet as compared to the 24% casein control diet. Protein malnutrition markedly affected the composition of acyl-linked fatty acids in the synaptic membranes. The increases in the ratio of 22:6 (n-3)/22:5 (n-6) fatty acids were especially compromised.
TL;DR: The results, together with some kinetic parameters, suggest that ethanolamine and choline phosphotransferases are affected differently by aging.
Abstract: The incorporation of cytidine-containing precursors (CDP-Cho and CDP-Etn) into the main phospholipid classes of cellular fractions enriched in neurons and glial cells from whole rat brains of different ages was examined. The rate of synthesis of choline phosphoglycerides in neuronal homogenates significantly decreased with age up to 18 months; after this time no additional decrease was found. The decrease of CDP-Etn incorporation in neurons was found to be less significantly affected by age up to 18 months, but the enzymic activity decreased after 18 months of age. No changes were found in the corresponding glial activity at any age. Biochemical phenomena that occur in 18-month-old rat brain (aged animals) were compared with phenomena occurring in 2-month-old rat brain (adult animals). No significant variations of lipid composition were found in neurons from either 18-month-old or 2-month-old rat brain. These results, together with some kinetic parameters, suggest that ethanolamine and choline phosphotransferases are affected differently by aging.
TL;DR: After hypoxic treatment the incorporation of the labeled precursors into DNA, RNA, and proteins was impaired to a different extent in the three brain regions and in the various subcellular fractions examined; DNA and RNA labeling in cerebellar mitochondria and protein labeling in microsomes of the threebrain regions examined were particularly affected.
Abstract: The incorporation of [methyl-3H]thymidine into DNA, of [5-3H]uridine into RNA, and of [1-14C]leucine into proteins of cerebral hemispheres, cerebellum, and brainstem of guinea pigs after 80 hr of hypoxic treatment was measured. Both in vivo (intraventricular administration of labeled precursors) and in vitro (tissue slices incubation) experiments were performed. The labeling of macromolecules extracted from the various subcellular fractions of the above-mentioned brain regions was also determined. After hypoxic treatment the incorporation of the labeled precursors into DNA, RNA, and proteins was impaired to a different extent in the three brain regions and in the various subcellular fractions examined; DNA and RNA labeling in cerebellar mitochondria and protein labeling in microsomes of the three brain regions examined were particularly affected.
TL;DR: Cerebral cortex and striatum of lithium-treated rats had a decreased apparent dissociation constant and a lower receptor concentration of naloxone binding sites, and Membranes obtained from the rats treated with lithium showed lower specific binding of both [3H]nalox one and [3 H]DHM.
Abstract: The effects of lithium and sodium were studied in the corpus striatum and cerebral cortex of rats. Lithium was inhibitory at low concentrations but at 20 mM it increased the binding of [G-3H]naloxone (specific activity 15.6 Ci/mmol). Sodium stimulated the high-affinity binding of this compound. Membranes obtained from the rats treated with lithium showed lower specific binding of both [3H]naloxone and [3H]DHM. Binding of [3H]d-alanine Leu-enkephalin was not changed in the brains of lithium-treated rats, but that of [3H]-spiroperidol was lowered. Cerebral cortex and striatum of lithium-treated rats had a decreased apparent dissociation constant and a lower receptor concentration of naloxone binding sites.
TL;DR: CDP-choline treatment was not able to reverse the effect of hypoxia on DNA labeling, but it was able to remove the effect from hypoxic treatment on RNA and protein labeling.
Abstract: The effect of CDP-choline on the in vivo incorporation of labeled precursors into DNA, RNA, and proteins in cerebral hemispheres, cerebellum, and brainstem of guinea pigs after hypoxic treatment was studied. The labeling of macromolecules extracted from the various subcellular fractions of these brain regions was also determined. Hypoxic treatment affected macromolecular labeling to a different extent in the three brain regions examined. CDP-choline treatment was not able to reverse the effect of hypoxia on DNA labeling, but it was able to remove the effect of hypoxia on RNA and protein labeling. The action of CDP-choline was particularly evident on the labeling of RNA in nuclei and mitochondria of the cerebellum and on the labeling of proteins in microsomes of the three brain regions examined.
TL;DR: The results confirm the conclusion that membrane fractions A and B are derived from the exposed synaptosome surface and show that GM1 is the major ganglioside species available for enzyme oxidation at the surface.
Abstract: Gangliosides in the external surface of intact synaptosomes from rat brain cortex have been studied by oxidation of exposed galactose and galactosamine groups with galactose oxidase followed by reduction with labeled sodium borohydride. Purified synaptosomes were labeled, disrupted by osmotic shock, and the particulate components fractionated on diatrizoate to give four synaptosomal membrane fractions (A-D) and a mitochondrial pellet (E). Fractions A and B represent synaptosomal plasma membranes. When intact synaptosomes were labeled, the major portion of the total radioactivity incorporated into ganglioside fraction was found to be in GM13 species. With isolated membrane fractions little selectivity was seen: (1) more label was present compared to intact synaptosomes, and (2) zones corresponding to GM2, GM1, GD1a, GD1b were the major gangliosides labeled. The results confirm the conclusion that membrane fractions A and B are derived from the exposed synaptosome surface and also show that GM1 is the major ganglioside species available for enzyme oxidation at the surface.
TL;DR: Relationships of TAG to selective mechanisms of phosphoglyceride synthesis were indicated and direct acyl exchange between TAG and phospholipids was not demonstrated.
Abstract: Metabolism of triacylglycerol (TAG) in developing brain has been examined. TAG is a relatively minor fraction of brain lipid in both suckling and adult rats and cannot be accounted for as entrapped blood. When glycerol tri[1-14C]oleate and [2-3H]glycerol trioleate were simultaneously injected intracerebrally into suckling rats, both labels appeared in diacylglycerol and the major phospholipids; acyl chain label was incorporated more extensively at early time points, with choline phosphoglycerides being most actively labeled. With [1-14C]fatty acids and [2-3H] glycerol administration, the specific activity of TAG was much greater than that of the more abundant phospholipids. Although direct acyl exchange between TAG and phospholipids was not demonstrated, relationships of TAG to selective mechanisms of phosphoglyceride synthesis were indicated.
TL;DR: In cerebellum the GSH/GSSG ratio was significantly decreased in the interictal stage of E1 mice (stimulated group), but in ddY mice this ratio was decreased before convulsions induced by pentylenetetrazol and during submaximal ECS.
Abstract: Concentration changes of reduced glutathione (GSH) and oxidized glutathione (GSSG) were studied by fluorometric assay witho-phthalaldehyde to clarify the relationship between seizure mechanism and the glutathione redox state. In cerebellum the GSH/GSSG ratio was significantly decreased in the interictal stage of E1 mice (stimulated group), but in ddY mice this ratio was decreased before convulsions induced by pentylenetetrazol and during submaximal ECS. No change was found in the GSH/GSSG ratio of the cerebellum during and after convulsions induced by pentylenetetrazol and maximal ECS. GSH levels in cerebrum in the interictal stage of E1 mice (stimulated group) were lower compared to control E1 mice. In ddY mice submaximal ECS increased GSSG levels in cerebrum so that the GSH/GSSG ratio was decreased.
TL;DR: After incubation at 37°C for 1 hr without added factors, the phospholipid degradation, as well as the appearance of free fatty acids, were higher in the ischemic samples (especially after 1 min of treament) as compared to controls.
Abstract: Synaptosomal phosphoglycerides were labeled after incubation with [1-14C]arachidonic acid, ATP, Mg2+, CoASH, and a small amount of 1-acylglycerophosphocholines Under this incubation system, radioactivity was directed largely to diacylglycerophosphocholines but diacylglycerophosphoinositols were also labeled to a lesser extent Synaptosomes obtained after a 5-min ischemic treatment indicated a decrease (10–20%) in incorporation of radioactivity into the phospholipids The ischemic synaptosomes also tended to retain a larger portion of the labeled arachidonate during the wash with bovine serum albumin Upon incubation of the prelabeled synaptosomes in a sucrose-Tris (pH 74) medium at 37°C, a time-dependent release of labeled arachidonate from the phospholipids was observed in both control and ischemic samples Arachidonate release from the prelabeled synaptosomes was not affected by EDTA (1 mM) or taurocholate (04%) but was stimulated by Ca2+ (25 mM) or Ca2+ (35 mM) together with EDTA (1 mM) After incubation at 37°C for 1 hr without added factors, the phospholipid degradation, as well as the appearance of free fatty acids, were higher in the ischemic samples (especially after 1 min of treament) as compared to controls
TL;DR: It is demonstrated that the β nerve growth factor will interact with various acidic proteins apparently nonspecifically, and this interaction may be the cause of the previously reported activation of the β nerves growth factor when bovine serum albumin is present in a typical bioassay.
Abstract: We have demonstrated that the β nerve growth factor will interact with various acidic proteins apparently nonspecifically. When125I-labeled β nerve growth factor at a concentration of 3.8×10−10 M is incubated with an acidic protein at 2 mg/ml (4.5×10−6–4.4×10−5 M), a complex is formed. This complex changes the isoelectric point of the125I-labeled β nerve growth factor sufficiently so that the125I-labeled β nerve growth factor migrates anomalously in polyacrylamide gel electrophoresis. The interaction between β nerve growth factor and bovine serum albumin, which appears to be complex, may be the cause of the previously reported activation of the β nerve growth factor when bovine serum albumin is present in a typical bioassay.
TL;DR: It is suggested that pipecolic acid is involved in either synaptic transmission or in its modulation at GABA synapses in the central nervous system.
Abstract: The active uptake of [3H]pipecolic acid increased with incubation time and its uptake at 3 min was half of that at 20 min. [14C]GABA uptake rose earlier, and its uptake at 3 min was almost 80% of that at 20 min. On the other hand, a ratio (pellet/medium) of [3H]pipecolic acid uptake into glial cell-enriched fractions, was much less (0.4–0.6) than that of [14C]GABA (25.8–74.1). GABA, 10−4 M, and pipecolic acid, 10−4 M, produced a significant inhibition of [3H]pipecolic acid uptake into P2 fractions. Pipecolic acid, 10−4 M, significantly reduced the synaptosomal and glial uptake of [14C]GABA. GABA, 10−4 M, affected neither spontaneous nor high K+-induced release of [3H]pipecolic acid from brain slices. It is suggested that pipecolic acid is involved in either synaptic transmission or in its modulation at GABA synapses in the central nervous system.
TL;DR: The present data together with results obtained previously with intracarotid injections suggest that pipecolic acid is taken up in the mouse brain from the circulation, and a small part is retained and probably metabolized by brain and kidney.
Abstract: The long-term accumulation of pipecolic acid, as well as its disappearance following exogenous administration was studied in brain and other organs of the mouse. Mice were pulse-injected intraperitoneally or intravenously with 1μCi[3H]D,l-pipecolic acid (6.9 nmol/mouse=2.9 μg/kg). The total radioactivity retained in tissues was measured in brain, liver, and kidney, as well as in plasma during the period 1 min to 24 hr. TLC separation of DNP-derivatives was performed. Three features of the pattern of retention of pipecolic acid are most salient; first the rapid accumulation in brain, second the rapid secretion of this compound in the urine, and third the long-lasting steady levels of radioactivity maintained in brain.
TL;DR: It is suggested that extracellular amino acids generally penetrate more readily into astrocytes than into neurons, and both cell types transport essential amino acids more effectively than other amino acids.
Abstract: The uptake of tritium-labeled L-leucine, L-lysine, L-aspartic acid, and glycine by neurons and astrocytes isolated from the cerebral cortex of 3-week-old rats was followed for varying periods up to 40 min at amino acid concentrations from 1 to 2000 micromol/liter in medium. The effects of a low-sodium (15.5 mmol/liter) medium on the uptake were also studied. The influx of the amino acids was faster into astrocytes than into neurons. Leucine penetrated into the cells faster than the other amino acids. Amino acids transport was mainly saturable at the lowest amino acid concentrations studied, whereas nonsaturable penetration into the cells dominated in the millimolar concentration range. The saturable transport comprised only one transport system with relatively small transport constants, resembling in nature the so-called high-affinity transport. The maximal velocities of transport were about two times higher in astrocytes than in neurons. In neurons the partial substitution of sodium by choline in medium had the most effect in reducing the influx of glycine and aspartic acid. In astrocytes the effects were generally less pronounced. The results suggest that extracellular amino acids generally penetrate more readily into astrocytes than into neurons. Both cell types transport essential amino acids more effectively than other amino acids.
TL;DR: The finding of oligoclonal IgG in EAE reveals yet another immunologic correlation between EAE and the human demyelinating disease, multiple sclerosis.
Abstract: Rabbits sensitized with whole nervous tissue or myelin basic protein (MBP) plus adjuvant and developing experimental allergic encephalomyelitis (EAE) were studied for the presence of oligoclonal immunoglobulin (Ig) bands in spinal fluid and serum. Samples obtained prior to sensitization and at the time of sacrifice were concentrated and subjected to agar gel electrophoresis. Of 11 rabbits receiving whole nervous tissue and developing severe clinical signs of EAE, 7 showed new oligoclonal Ig bands in spinal fluid and in serum obtained 19 days or more after sensitization. With MBP sensitization, 2 of 6 rabbits exhibited new spinal fluid bands, while all 6 rabbits studied demonstrated serum banding. The bands were identified as IgG by immunochemical studies using peroxidase-labeled antisera and byStaph. protein A absorption. The majority of animals showed no banding in presensitization samples. The finding of oligoclonal IgG in EAE reveals yet another immunologic correlation between EAE and the human demyelinating disease, multiple sclerosis.
TL;DR: Evidence is presented that pipecolic acid is synthesized both in vitro and in vivo in the mouse brain, actively transported in vivo into the brain, and taken up in vitro by synaptosomal preparations.
Abstract: The uptake of pipecolic acid by the mouse brain was compared to that of several amino acids and amines, following an injection of a double-labeled mixture into the carotid artery. In general, BUI (brain uptake index) values were lower in the mouse than those previously reported in the rat. The only exception was proline. Lysine, a precursor of pipecolic acid biosynthesis in brain, showed a higher BUI than pipecolic acid. The BUI ofD,l-[3H]pipecolic acid was found to be 3.39 (at 0.114 mM). This was saturable between a concentration of 0.114 and 3.44 mM. Kinetic analysis suggests the presence of two kinds of transport systems. Substances structurally related to pipecolic acid, such as nipecotic acid, isonipecotic acid,l-proline, and piperidine show a significant inhibitory effect. Among the amino acids tested, only GABA showed an inhibitory effect. Data are reported which, when considered with other findings (5), present evidence that pipecolic acid is (1) synthesized both in vitro and in vivo in the mouse brain, (2) actively transported in vivo into the brain, and (3) taken up in vitro by synaptosomal preparations.
TL;DR: The pattern of the synthesized proteins underwent considerable alteration with age in young cultures in which the total content of protein was still increasing, but it was remarkably stable after the age of two weeks, suggesting that a very considerable part of the protein synthesis in the adult brain may take place in astrocytes.
Abstract: Protein synthesis, measured as leucine incorporation into acid-precipitable proteins, was determined in astrocytes in primary cultures obtained from the cerebral hemispheres of newborn mice. As can be expected for eucaryotic, ribosomal protein synthesis, the incorporation was almost completely inhibited by cycloheximide (0.01 mM), but unaffected by chloramphenicol (0.03 mM). The rate of synthesis, measured during exposure to a high (0.8 mM) concentration of leucine was 5.4 nmol/hr/mg protein in mature (i.e., at least 4-week-old) cultures. This value is at least twice as high as the protein synthesis rates reported for the adult brain in vivo, suggesting that a very considerable part of the protein synthesis in the adult brain may take place in astrocytes. The molecular weight distribution of the synthesized proteins was determined by polyacrylamide gel electrophoresis, demonstrating synthesis of at least 50 different polypeptides, ranging in molecular weight between 190,000 and 27,000 daltons. The pattern of the synthesized proteins underwent considerable alteration with age in young cultures in which the total content of protein was still increasing, but it was remarkably stable after the age of two weeks. Exposure to dibutyryl cyclic AMP, which is known to alter morphology, content of glial fibrillary acidic protein (GFA), and activities of certain enzymes in the cultures in the cultured astrocytes, caused marked alterations in the pattern of the synthesized proteins.
TL;DR: The most dense axolemma-enriched fraction is over fourfold enriched in glyco-protein content compared with myelin, with at least 10 different molecular-weight classes of glycoproteins as identified by Schiff stain of polyacrylamide gel protein profiles.
Abstract: Axolemma-enriched fractions were isolated from the white matter of bovine corpus callosum via a purified preparation of myelinated axons which were osmotically shocked and fractionated on a discontinuous density gradient. Two membrane fractions of differing density were obtained; both were somewhat enriched over white matter whole homogenate in specific activity of acetylcholinesterase and 5′-nucleotidase and maximal binding capacity for saxitoxin. Both membrane fractions contained appreciable amounts of 2′, 3′-cyclic nucleotide 3′-phospho-hydrolase; the specific activity of antimycin-sensitive NAPH-cytochromec reductase and cytochromec oxidase indicated low levels of contamination by microsomal and mitochondrial membrane. The myelin which is concomitantly isolated with the axolemma-enriched fractions has a lipid and protein composition comparable to that of myelin isolated by other procedures. Both axolemma-enriched fractions contain about one half of their dry weight as lipid comprised of approximately 25% cholesterol, 25% galactolipid (cerebrosides and sulfatides in a molar ratio of about 4:1) and 50% phospholipid, mostly choline phosphatides and ethanolamine phospholes in an equimolar ratio. The axolemma fractions are also enriched in ganglioside content relative to the myelin fraction. The polypeptides of the axolemma-enriched fractions range from 20,000 to over 200,000 in molecular weight; the predominant proteins are in the range from 50,000 to 69,000. The most dense axolemma-enriched fraction is over fourfold enriched in glyco-protein content compared with myelin, with at least 10 different molecular-weight classes of glycoproteins as identified by Schiff stain of polyacrylamide gel protein profiles. The differences and similarities in the molecular composition of axolemma-enriched preparations which have been characterized to date are discussed.
TL;DR: Rat brain microsomal phosphatidylinositol kinase activity was maximally activated in the presence of either 3 mM sodium deoxycholate, 2% Triton-X-100, or 30–40 mM octylglucoside, and this activity may have disrupted interactions of the enzyme with other hydrophobic proteins sufficiently to allow its substantial purification by conventional or affinity chromatography techniques.
Abstract: Rat brain microsomal phosphatidylinositol kinase activity was maximally activated in the presence of either 3 mM sodium deoxycholate, 2% Triton-X-100, or 30–40 mM octylglucoside. Among these detergents, 1% Triton-X-100 was most effective in solubilizing the enzyme, and after treatment with, this agent, 100% of the activity was recovered in the high speed supernatant. Octylglucoside solubilized 40% of the enzyme at concentrations below its critical micelle concentration of 25 mM and up to 80% at higher levels. Solubilized phosphatidylinositol kinase failed to adsorb to adenosine nucleotide affinity resins. However, when the Triton-X-100 extract was chromatographed on an uncharged hydrophobic resin, consisting of dodecyl chains attached to Sepharose 4B by ether bonds, nearly all the enzyme activity was retained, and from 44–85% could be eluted with 8 mM sodium deoxycholate. Solubilization followed by hydrophobic chromatography resulted in several-fold purification of phosphatidylinositol kinase and may have disrupted interactions of the enzyme with other hydrophobic proteins sufficiently to allow its substantial purification by conventional or affinity chromatography techniques.