bluuebunny/crossref_metadata_2025 huggingface dataset:
RIBOSOMAL SUBUNITS FROM NEONATAL MOUSE BRAIN HIGHLY ACTIVE IN POLYPHENYLALANINE SYNTHESIS
Authors: van der Saag Paul T. , de Greeff Theo F. J.
Published: October 2006

Abstract— A highly active subcellular protein synthesising system is described, in which uncomplexed ribosomes isolated from 5 to 7 day old mouse brain can be reprogrammed with polyuridylic acid. Either purified free polyribosomes or microsomes were used as the starting material for the preparation of uncomplexed ribosomes by treatment with 0.5m‐KCl and puromycin. After reduction of the salt concentration 80S ribosomes were isolated by washing through sucrose. When, subsequently, zonal centrifugation in equivolumetric sucrose gradients containing 0.5 m‐KCI was performed, purified ribosomal subunits were obtained. Cross‐contamination of subunits was less than 5%. Re‐associated ribosomes and recombined isolated ribosomal subunits both showed high activities in vitro. Incorporation levels of 50–60 phenylalanine residues per ribosome could be reached, at a rate of 0.5–2.0 residues/min/ribosome, depending on the activity of the high speed supernatant enzymes added. It was shown by paper chromatography of the cell‐free product that only oligophenylalanine formation takes place. It was estimated that 6&70% of the ribosomes present in vitro were actively participating in the protein synthesis process.
CHEMICAL SYNTHESIS OF [3H]CEREBROSIDE [35S]SULFATE AND ITS IN VIVO METABOLISM IN RAT BRAIN
Authors: Nonaka Genichiro , Kishimoto Yasuo
Published: October 2006

—Double‐labeled sulfatide containing [3‐3H]lignoceric acid and [35S]sulfate was synthesized and injected intracerebrally into 28‐day‐old rats. The 3H‐labeled sulfatide was synthesized by condensing (RS)‐[3‐3H]lignoceroyl chloride with lysosulfatide which had been obtained by saponification of sulfatide. The 35S‐labeled sulfatide was synthesized by using [35S]sulfuric acid for sulfating 2′, 4′, 6′‐tri‐benzoyl‐galactosyl N‐fatty acyl, N‐benzoyl‐3‐0‐benzoyl‐sphingosine, which had been obtained by per‐benzoylation followed by solvolysis of calf brain nonhydroxycerebrosides. The perbenzoylated [35S]sul‐fatide was then subjected to mild alkaline saponification. Eight hours following the injection, the brain lipids contained various radioactive sphingolipids in addition to sulfatides. Fourteen per cent of the injected 3H was recovered in total lipids, and 26% of this was found in sulfatide. Nonhydroxy‐ and hydroxyceramides, nonhydroxy‐ and hydroxycerebrosides, and polar lipids contained 7, 1, 8, 3, and 22 per cent of the 3H found in total lipids, respectively. On the other hand, only 6% of the 35S injected was recovered in total lipids; 63% of this was found in sulfatide, 5% in a mixture of seminolipid and cholesterol sulfate and 10% in a water‐soluble material.
A ROLE FOR MICROTUBULES IN THE REGULATION OF 3‐HYDROXY‐3‐METHYLGLUTARYL COENZYME A REDUCTASE AND CHOLESTEROL BIOSYNTHESIS IN CULTURED GLIAL CELLS 1
Authors: VOLPE J. J.
Published: October 2006

—Cultured C‐6 glial cells were utilized to evaluate the effect of the antimicrotubular drug, Colcemid, on 3‐hydroxy‐3‐melhylglutaryl coenzyme A (HMG‐CoA) reductase and cholesterol synthesis in cultured C‐6 glial cells. The data indicate that Colcemid causes a marked inhibition of cholesterol synthesis (from [14C]acetate or 3H2O) in these cells. A concentration of 0.5 μM led to a 50% lower rate of synthesis after 2 h and an 80–85% lower rate after 12 h or longer. That the effect of Colcemid is mediated at the level of HMG‐CoA reductase was shown by defining closely coordinate temporal and quantitative changes in the activity of this enzyme under identical conditions. No comparable change in cell growth or in total protein synthesis accompanied the effect of Colcemid. The drug did lead to a decrease in the rate of DNA synthesis (from [3H]thymidine) but this effect was preceded by the decrease in the rate of cholesterol synthesis. Marked changes in glial cell shape were induced by exposure to Colcemid, and the temporal and quantitative aspects of these changes appeared to closely parallel the effects on reductase activily and cholesterol synthesis.

The dala suggest that microtubules are involved in the regulation of HMG‐CoA reductase and cholesterol synthesis in mammalian cells and that there are important interrelations between microtubules, glial differentiation and cholesterol synthesis.
ADDENDUM TO A NEW MATHEMATICAL APPROACH TO METABOLISM OF [14C]GLUCOSE: THE PYRUVATE CARBOXYLASE REACTION 1
Authors: Larrabee Martin G.
Published: October 2006

—Previously published equations for analysis of [14C]glucose metabolism assumed that products of glycolysis enter the citric acid cycle only through acetyl‐CoA (Larrabee, 1978). These equations are now extended to include entrance into the citric acid cycle through the pyruvate carboxy‐lase reaction as well as via acetyl‐CoA and are applied to previously reported data from dorsal root ganglia of 15‐day‐old chicken embryos. The rate of output of labelled CO2 in the presence of [2‐14C] glucose could not be accounted for if the flux rate into the citric acid cycle through the pyruvate carboxylase reaction was assumed to be more than about 10–15% of that through acetyl‐CoA. It is concluded (1) that the pyruvate carboxylase reaction is a relatively minor source of material for the citric acid cycle in these ganglia and (2) that the previous conclusions about [14C]glucose metabolism, which ignored the pyruvate carboxylase reaction, need not be modified in the light of this reanalysis.
ALTERATIONS IN THE BINDING OF [3H]LEUCINE‐ENKEPHALIN TO STRIATAL MEMBRANES BY ENDOGENOUS FACTORS
Authors: Rothman Richard B. , Westfall Thomas C.
Published: October 2006

—Saturation binding studies with [3H]leu‐enk ([tyrosyl‐3, 5‐3H(N)]5leu‐enkephalin) revealed the presence of high and low affinity binding sites in a paniculate fraction derived from rat striatum. The binding of [3H]leu‐enk to the high affinity component (KD= 2.0 ± 0.3 nM) was sensitive to morphine and levorphanol, while the binding to the low affinity component (KD= 21 ± 2 nM) was not.

Incubation of the membranes, prior to assay for 30 min at 37°C, followed by centrifugation at 27, 000 g for 20 min in order to pellet the membranes allowed the detection of a factor, present in the high speed supernatant, which caused a dose‐dependent inhibition of the binding of [3H]leu‐enk to the morphine‐sensitive and insensitive binding components.

Investigations into the nature of the morphine‐insensitive binding component demonstrated that it was an artifact since it was not detectable when bound and free ligand were separated by centrifugation. Furthermore, [3H]leu‐enk bound to Whatman glass fiber filters, used to collect bound ligand, in a morphine‐insensitive manner, and under conditions where the binding of [3H]leu‐enk to the morphine sensitive component diluted proportionally with serial dilutions of the membranes, the binding to the morphine‐insensitive component did not.

The factor present in the high speed supernatant did not dialyze and its effects were mimicked by either trypsin or soybean trypsin inhibitor, but not by bovine serum albumin.

The apparent inhibition of the binding of [3H]leu‐enk to these binding components is probably not of biological significance, but the fact that the artifactual morphine insensitive binding component of striatal membranes has been shown to decrease by 20–30% following lesions of the substantia nigra suggests that the influence of this endogenous factor must be controlled for.
ATP‐Dependent Ca2+ Accumulation by Microvesicles Isolated from Bovine Neurohypophyses
Authors: Torp‐Pedersen Christian , Særmark Torben , Bundgaard Magnus , Thorn Niels A.
Published: October 2006

: Microvesicles with and without coats were isolated from bovine neurohypophyses by a series of ultracentrifugation steps. They were similar to microvesicles previously isolated from brain. In the presence of ATP and Mg2+, the microvesicles accumulated calcium. Oxalate stimulated and the Ca2+ ionophore A23187 inhibited calcium accumulation. In the presence of Ca2+ and Mg2+ the microvesicles also demonstrated ATPase activity. As judged by SDS electrophoresis in polyacrylamide gels microvesicle protein composition was very different from that of neurosecretory granules, suggesting that they are not granule membranes “retrieved” after exocytosis. They may have an important function in regulation of Ca24 ion concentration.
Evidence for the Presence of CDP‐Ethanolamine: 1,2‐diacyl‐sn‐glycerol Ethanolaminephosphotransferase in Rat Central Nervous System Myelin
Authors: Wu Po‐Shun , Ledeen Robert W.
Published: October 2006

: Highly purified rat brain myelin isolated by two different procedures showed appreciable activity for CDP‐ethanolamine: 1,2‐diacyl‐sn‐glycerol ethanolaminephosphotransferase (EC 2.7.8.1). Specific activity was close to that of total homogenate and approximately 12–16% that of brain microsomes. Three other lipid‐synthesizing enzymes, cerebroside sulfotransferase, lactosylceramide sialyltransferase, and serine phospholipid exchange enzyme, were found to have less than 0.5% the specific activity in myelin compared with microsomes. Washing the myelin with buffered salt or taurocholate did not remove the phosphotransferase, but activity was lost from both myelin and microsomes by treatment with Triton X‐100. It resembled the microsomal enzyme in having a pH optimum of 8.5 and a requirement for Mn2+ and detergent, but differed in showing no enhancement with EGTA. The diolein Km was similar for the two membranes (2.5–4 × 10−4m), but the CDP‐ethanolamine Km was lower for myelin (3–4 × 10−5m) than for microsomes (11–13 × 10−5m). Evidence is reviewed that this enzyme is able to utilize substrate from the axon in situ.
An Apparent Paradox in the Occurrence, and the In Vivo Turnover, of C‐Terminal Tyrosine in Membrane‐Bound Tubulin of Brain
Authors: Nath Jayasree , Flavin Martin
Published: October 2006

: Tubulin tyrosine ligase catalyzes the reversible addition of tyrosine to the C‐terminus of tubulin α chains. By using ligase and carboxypeptidase A in conjunction, we have previously shown that brain cytoplasmic tubulin exists in three forms: 15–40% already has C‐terminal tyrosine, another 10‐30% can accept additional tyrosine, and about one‐half is an uncharacterized species which is not a ligase substrate. A membrane‐bound fraction of brain tubulin, purified by vinblastine precipitation from a detergent extract, has been found to differ by the complete absence of preexisting tyrosine. The membrane fraction from which tubulin was extracted also contained masked forms of both ligase and a distinct detyrosylating enzyme, which can be released by detergent extraction. The turnover of α‐chain C‐terminal tyrosine in vivo was studied by incubating brain mince with labeled tyrosine, or injecting it intracerebrally, under conditions where protein synthesis was inhibited. Tyrosine appeared to turn over to about the same extent in membrane‐bound, as in soluble, tubulin. This apparently paradoxical result was not due to ATPase in the membrane fraction, which might have allowed ligase‐catalyzed exchange between free and fixed tyrosine. Authentic [14C]tyrosylated tubulin added to the brain membrane fraction was not detyrosylated or subject to endoprotease digestion during subsequent procedures to isolate tubulin. The unexpected finding that tubulin tyrosylated at the C‐terminal in vivo appears to be in the “non‐substrate” fraction points toward a possible resolution of the paradox.
Inhibition of [3H]Spiroperidol Binding by In Vitro Addition of Ethanol
Authors: Hruska Robert E. , Silbergeld Ellen K.
Published: October 2006

: The in vitro addition of ethanol at concentrations greater than 0.2% produced an inhibition of [3H]spiroperidol binding. This inhibition was competitive, lowering the affinity without altering the maximum number of sites. Both the rates of association and dissociation were altered such that the ligand spent less time attached to the receptor. Since the binding was competitive, little inhibition was seen at ligand concentrations greater than 300 pm. These results are important for in vitro studies when drugs are dissolved in ethanol, and also for in vivo studies on the effects of ethanol.
Screening for Rabbit Brain Neuropeptide‐metabolizing Peptidases. Inhibition of Endopeptidase B by Bradykinin Potentiating Peptide 9a (SQ 20881)
Authors: Martins Antonio Roberto , Caldo Hildeberto , Luna Coelho Helena Lutéscia , Moreira Ayrton Custódio , Antunes‐Rodrigues Jose , Greenet Lewis Joel , de Camargo Antonio Carlos Martins
Published: October 2006

Abstract: Neutral thiol‐activated peptidases present in the pH 5‐soluble fraction of rabbit brain (separated by step‐elution chromatography on diethylaminoethyl cellulose) were screened for the hydrolysis of bradykinin, Lysbradykinin, Met‐Lys‐bradykinin, angiotensin I, angiotensin II, substance P, luteinizing hormone‐releasing hormone (LH‐RH) and neurotensin by bioassay. The column effluent was monitored for bradykinin inactivation and arylamidase activity and combined in six pools on the basis of bradykinin inactivation. The pools were characterized by determining the peptide fragments and amino acids released from bradykinin with an amino acid analyzer. Pools 1 through 3 contained 80% of the kininase activity and essentially all of the endopeptidase A and B activity, whereas pools 4 through 6 accounted for 98% of the recovered arylamidase activity. Bradykinin, angiotensin I, angiotensin II and substance P were inactivated by all the pools, whereas LH‐RH and neurotensin were inactivated by pools 3 and 4 and pools 3, 4 and 5, respectively. These data show that rabbit brain contains peptidases having some selectivity for the inactivation of neuropeptides. Endopeptidase B purified from pool 3 is inhibited by bradykinin‐potentiating peptide 9a (BPP9a' SQ 20881) (Glu‐Trp‐Pro‐Arg‐Pro‐Gln‐Ile‐Pro‐Pro), a competitive inhibitor of the hydrolysis of bradykinin (Km= 3.5 ± 10−5m, Ki= 3 ± 10−6m) which also completely inhibits the inactivation of LH‐RH.
Tubulinyl‐tyrosine Carboxypeptidase from Chicken Brain: Properties and Partial Purification
Authors: Argarana C. E. , Barra H. S. , Caputto R.
Published: October 2006

Abstract: Tyrosine can be released from tubulinyl‐tyrosine by the action of a brain carboxypeptidase. The molecular weight of this enzyme found by gel filtration through a column of Sephadex G‐200 was 90,000. The enzyme was very unstable in a purified preparation in which the activity per milligram of protein was increased 250‐fold with respect to the starting material. The precise magnitude of the purification cannot be stated because of the unknown amount of endogenous tubulinyl‐tyrosine in the material to be assayed. A comparative study was done between tubulinyl‐tyrosine carboxypeptidase (TTCP) activity and pancreatic carboxypeptidase A (CPA, EC 3.4.12.2) activity using tubulinyl‐[14C]tyrosine as substrate. The most remarkable differences found are: MgCl2 (2 mM), phenyl acetate (10 mM), or EDTA (5 mM) increased the TTCP activity whereas the CPA activity was strongly inhibited by these compounds, lodoacetate (2 mM) and ZnCl2 (0.1 mM) inhibited the TTCP activity more than the CPA activity. Contrarily, mercaptoethanol (50 mM) and dimethyl sulfoxide (5%) showed a stronger inhibitory effect on CPA than on TTCP. Of several N‐carbobenzoxy dipeptides (Z‐dipeptides) tested the greatest inhibitory effects on TTCP activity were obtained with Z‐Glu‐Tyr and Z‐Glu‐Phe, although strong inhibitory effects on CPA were also obtained with other Z‐dipeptides.
Human Brain Cerebroside β‐Galactosidase: Deficiency of Transgalactosidic Activity in Krabbe's Disease
Authors: Carter Thomas P. , Beblowski Diane W. , Savage Michael H. , Kanfer Julian N.
Published: October 2006

Abstract: Under experimental conditions optimal for the assay of D‐galactosyl‐N‐acylsphingosine galactohydrolase (EC 3.2.1.46) activity, homog‐enates of neurologically normal human brain tissue could transfer galactose from galactosyl ceramide (gal‐cer), lactosyl ceramide (lac‐cer), 4‐methylumbelliferyl‐ β‐galactoside (4‐MU‐gal), or p‐nitrophenyl‐ β‐galactoside (PNP‐gal) to [1‐14C]oleoyl sphingosine, but homogenates of brain tissue from patients with Krabbe's disease lacked this ability. The rate of hydrolysis of ganglioside GM1 and to a lesser extent, of PNP‐gal by homogenates of Krabbe's brain tissue was also decreased. Activity of PNP‐ β‐galactosidase in normal brain tissue, like that of cerebroside β‐galactosidase from the same source, was considerably more heat‐stable than the activity of either 4‐MU‐ β‐galactosidase or the predominant GM1β‐D‐galactosidase (EC 3.2.1.23). Lac‐cer and GM1, as well as 4‐MU‐gal and PNP‐gal, were competitive inhibitors of human‐brain cerebroside β‐galactosidase. These findings confirm the ability of mammalian cerebroside β‐galactosidase to catalyze a transgalactosylation reaction and provide additional information on the substrate specificity of human brain cerebroside β‐galactosidase.
Nucleocytoplasmic Transport of RNA and Protein Synthesis in Developing Brain from Normal and Undernourished Rats
Authors: Duvilanski Beatriz H. , de Guglielmone Alicia E. Ramirez , Gomez Carlos J.
Published: October 2006

We have reported earlier that neonatal undernutrition in the rat produces a transient decrease in brain nuclear RNA synthesis, but a marked and permanent increase in the labeling ratio of nuclear to microsomal RNA following a 1‐h in vivo pulse of labeled precursor (Ramirez de Guglielmone et al., 1974). Although these findings would suggest that posttranscriptional processing and transport of RNA transcripts are permanently affected by this condition, it is also possible that the observed alterations could be due to changes in cytoplasmic RNA metabolism.

We report here the effect of neonatal undernutrition on nucleocytoplasmic transport of messenger and ribosomal RNA in a cell‐free system that precludes detectable nuclear lysis. We also attempt to relate alterations in transport to polysomal aggregation and protein synthesis in viva.
Hydrolysis of Galactose from Glycolipids and Glycoprotein by Young Rat Brain β‐Galactosidases Immobilized to Sepharose 4B
Authors: Yeung Kwok‐Kam , McKinney Richard A. , Dain Joel A.
Published: October 2006

An extract of glycosidic enzymes from young rat brain was immobilized to cyanogen bromide‐activated Sepharose 4B. Most glycosidases retained approximately 10‐25% of their activities after immobilization. Immobilized β‐galactosidases were used repeatedly without detectable loss of enzyme activity in the hydrolysis of p‐nitrophenyl‐β‐d‐galactopyranoside. In addition to the synthetic substrate, the immobilized rat brain β‐galactosidases could also hydrolyze galactose from lactose, galactosylcerebroside, asialofetuin, and GM1‐ganglioside. The hydrolysis of GM1‐ to GM2‐ganglioside was confirmed on TLC.
Axonal Transport of [35S]Methionine‐Labeled Proteins in Two Intra‐Brain Tracts of the Rat
Authors: Padilla Stephanie S. , Morell Pierre
Published: October 2006

[35S]Methionine was stereotaxically injected into the dorsalateral geniculate body (DLGB) of adult male rats, and 1 h to 10 days post‐injection the DLGB and projection site (striate cortex) were dissected out and solubilized in 1% sodium dodecyl sulfate. Samples were analyzed for acid‐precipitable radioactivity, and radioactivity in different molecular weight classes was determined following discontinuous gel electrophoresis on both tube and slab gels. Acid‐precipitable radioactivity in the DLGB peaked by 4 h and then declined over the time period studied. The molecular weight distribution pattern was complex and did not change appreciably with time. Radioactivity in the striate cortex arrived in at least three waves: rapidly transported proteins arrived between 2 and 4 h; a second wave of transport began to arrive at about 7 h post‐injection and there was a slight rise in specific activity for 2 days; finally, at 3 days post‐injection, there was a steep increase with the arrival of the bulk of the transported material. The electrophoretic distribution pattern of proteins arriving in the first wave included 40–50 identifiable bands ranging in molecular weight from 13,000 to 200,000. Of particular interest was a radioactive band of apparent molecular weight of 110,000, which was prominent at 4 h, but by 12 h showed very little labeling. The second wave of radioactivity contained primarily proteins of molecular weight classes already present, although there were quantitative differences. Several proteins in the molecular weight range of 43,000 to 78,000 were identifiable as characteristic of the third wave of transported material. Results from a study following injection of a hippocampus were similar: the electrophoretic distribution pattern of radioactive proteins extracted from the injected hippocampus resembled that of the DLGB, and also did not vary appreciably with time, while radioactive proteins in the contralateral hippocampus had an electrophoretic distribution pattern similar to that of the striate cortex and changed with time in a similar manner.
Density Profile and Basic Protein Measurements in the Myelin Range of Particulate Material from Normal Developing Mouse Brain and from Neurological Mutants (Jimpy; Quaking; Trembler; Shiverer and its mid Allele) Obtained by Zonal Centrifugation
Authors: Bourre J. M. , Jacque C. , Delassalle A. , Nguyen‐Legros J. , Dumont O. , Lachapelle F. , Raoul M. , Alvarez C. , Baumann N.
Published: October 2006

The particulate material of aquaeous homogenate of forebrain was separated by zonal centrifugation in a continuous 0.4‐1.2 m‐sucrose gradient after sedimentation at 100,000 g to eliminate the soluble material. Based on the absorbance at 280 nm, four major peaks were obtained from adult normal mice corresponding to 1.1 m (A), 0.68 m (B), 0.35 m (C), and 0.12 m (D) sucrose. The two smaller and lighter peaks, C and D, were not present when purified myelin was separated by the same procedure. B consisted of pure compact myelin; A was made of vesicles, sometimes with a double membrane. Throughout development, the myelin peak shifted from 0.58 m in young animals to 0.70 m in very old ones. Moreover, the myelin peak B drastically increased during development, as compared with peak A.‐In the Trembler, the profile was close to normal, with a slightly higher yield of myelin which also peaked at a higher density. In the quaking, there were only two shoulders in the myelin density range at 0.68 m and 0.75 m; in the Jimpy only a faint shoulder was seen, at approximatively 0.67 m. In the shiverer, B was absent and only an A peak was present, at approximatively 0.85‐0.90 m, which contained non‐compact lamellar membranes and myelin figures with an abnormal major dense line. In the mid (an allele of shiverer) the density profile resembled the one obtained in the shiverer (one peak in the 0.88 m region). When considering myelin basic protein (MBP) content in the normal developing animal, it was minimum in fraction A, mainly found in the B myelin peak, but also present in the light fraction (C + D). In young animals this latter peak was prominent, in contrast to the adult. In the Trembler mutant, the profile was close to normal; in the quaking, MBP was mainly found in peak A (0.85 m), and in the Jimpy MBP was very low and nearly constant throughout the gradient (with faint quantities in light C + D fraction). In mid, the content was very low, with a peak in the 0.88 m region, in contrast with its shiverer allele, where MBP is hardly detected. More meaningful myelin studies can be carried out by using zonal centrifugation in continuous sucrose gradient, to determine the density of mutant myelin and the degree of myelin maturation in animals.
Presynaptic Membrane Protein Phosphorylation Modulates the Release of GABA from Preloaded Synaptosomes
Authors: Brennan M. J. W. , Cantrill R. C.
Published: October 2006

Incubation of rat cortical synaptosomes in the presence of 1 mM ATP markedly reduced Ca2+‐dependent K+ ‐induced release of preloaded 3H‐GABA. This effect is probably dependent on phosphorylation of presynaptic membrane proteins. By contrast, the uptake of 3H‐GABA by the nerve endings was not significantly affected by phosphorylation. These data suggest that membrane protein phosphorylation and dephosphorylation may form a mechanism for the presynaptic control of transmitter release.
Chronic Alcohol Intake and Some Monogalactosyl Glycolipids of the Rat Brain
Authors: Vrbaški Snežana R. , Petrovió Gordana T. , Ristié Milosav S.
Published: October 2006

The effect on monogalactosyl glycolipid complement (cerebrosides with hydroxy and non‐hydroxy fatty acids, sulphatides and monogalactosyldiglyceride) after 38 weeks of feeding 25% sucrose‐32% ethyl alcohol have been studied. Chronic alcohol intake produced a statistically significant increase of cerebrosides with hydroxy and non‐hydroxy fatty acids and monogalactosyldi‐glyceride as well as decrease of sulphatides in male Wistar rats brain.
Regional Distribution of Neurotransmitter Synthesizing Enzymes in the Basal Ganglia of Human Brain
Authors: Gaspar P. , Javoy‐Agid F. , Ploska A. , Agid Y.
Published: October 2006

Tyrosine hydroxylase (TH, EC 1.14.16.2), glutamate decarboxylase (GAD, EC 4.1.1.15), and choline acetyltransferase (ChAT, EC 2.3.1.6), activities were used as markers of the presence of catecholaminergic, GABAergic, and cholinergic neurons in human brain. The disparity in the absolute values obtained in control caudate nucleus led us to study the pattern of distribution of these enzymes in given brain regions, with the idea that the pattern might be altered specifically in pathological conditions. The tridimensional distribution of TH, GAD, and ChAT activities was investigated within the caudate nucleus, the putarnen, and the pallidurn. In control patients, opposite rostro‐caudal and rnedio‐lateral gradients appeared for GAD and CAT; ChAT activity was higher at the caudal level and GAD activity was higher at the rostral level of caudate nucleus and putamen. In the medio‐lateral extent of putarnen and pallidurn, ChAT activity was highest in the lateral part of the putarnen; GAD was highest in the medial segment of the pallidum. Only GAD presented a particular dorsoventral pattern, the enzyme activity being highest in the ventral part of the caudate nucleus and the putarnen. No reproducible distribution was observed for TH. In Parkinsonian patients, a decrease of TH and GAD activities with abolition of gradients was observed, whereas the ChAT activity and pattern were not modified.
Phosphorylation of Synaptic Membranes
Authors: Berman Robert F. , Hullihan John P. , Kinnier William J. , Wilson John Eric
Published: October 2006

In vivo and in vitro phosphorylation of synaptic membrane proteins were compared. In vivo phosphorylation was carried out by injecting rats intraventricularly with 1 mCi of 32Pi‐orthophosphate. After a 40‐min isotope incorporation period, rats were decapitated and synaptic membranes isolated. In vitro phosphorylation was accomplished by incubating unlabeled synaptic membranes, isolated from noninjected rats, with 5 μM‐[γ‐32P]ATP. In vivo and in vitro32P‐labeled synaptic membranes were then fractionated electrophoretically on an SDS‐polyacrylamide (7.5%) slab gel. The Coomassie blue protein patterns for in vivo and in vitro32P‐labeled synaptic membranes were identical. In contrast, autoradiographs of these gels showed striking differences in the pattern of phosphate incorporation. Cyclic‐AMP (10 μM) maximally stimulated) the in vitro phosphate labeling of two protein bands (80,000 and 55,000 apparent molecular weight) which were not substantially labeled by the in vivo procedure.
Monoamine and Tissue Fluid Levels in Contused Spinal Cord of Sheep
Authors: Hinwood Barry G. , Yeo John D. , Kidman Antony D.
Published: October 2006

Methods are described for the determination of sheep spinal cord tissue fluid content and norepinephrine, serotonin, and dopamine concentrations after experimental injury. The amount of tissue fluid varied in different regions of sheep spinal cord following injury. Norepinephrine and serotonin wet weight concentrations were corrected for this variation in tissue fluid. Corrected norepinephrine wet weight cord concentrations did not change up to 3 hr after injury. Levels of serotonin at 60 min after injury were similar to controls. Dopamine was not detected in sheep spinal cord. α‐Methyl tyrosine significantly reduced fluid in the spinal cord at 75 min after injury. Hinwood B. G. et al. Monoamine and tissue fluid levels in contused spinal cord of sheep. J. Neurochem.35, 786–791 (1980).
Enzymatic Glucosylation of Dolichyl Monophosphate Formed Via Cytidine Triphosphate in Calf Brain Membranes
Authors: Scher Malka G. , Burton Willis Anne , Waechter C. J.
Published: October 2006

When calf brain membrane preparations containing endogenous dolichyl [32P]monophosphate (Dol‐32P), prelabeled enzymatically by [γ‐32P]‐CTP, are incubated with unlabeled UDP‐glucose, the formation of a mild acid‐labile [32P]phosphoglucolipid is observed. The biosynthesis of the [32P]phosphoglucolipid is dependent on the concentration of UDP‐glucose added, and no [32P]phosphoglycolipid appeared when UDP‐glucose was replaced by ADP‐glucose, UDP‐xylose, UDP‐galactose, UDP‐mannose, or UDP‐glucuronic acid. The 32P‐labeled product formed by the UDP‐glucose‐dependent reaction is chemically and chromatographically identical to glucosylphosphoryldolichol. Several enzymatic parameters of the glucosylation of the specific pool of Dol‐P, synthesized by the CTP‐mediated kinase, and the total available pool of Dol‐P have been compared by a double‐label assay utilizing endogenous, prelabeled Dol‐32P and UDP‐[3H]glucose as substrates.
Cell‐free Synthesis of the Myelin Basic Proteins in a Wheat Germ System Programmed with Brain Messenger RNA
Authors: Matthees Joanne , Campagnoni Anthony T.
Published: October 2006

Poly A(+) messenger RNA (mRNA) was isolated from the brains of 3‐week‐old mice and translated in a cell‐free system derived from wheat germ. Maximal stimulation of the system by brain mRNA was observed at a relatively low K+ concentration (45 mm) and low mRNA concentration (1–10 μg/ml). The translational system was dependent on an energy‐generating system and stimulated by the addition of spermidine and transfer RNA. Under optimal conditions, incorporation was linear for almost 45 min, but the overall stimulation with brain mRNA was relatively low (about twofold). In spite of the low stimulation, analysis of the translation products indicated that in the presence of brain mRNA polypeptides which co‐chromatographed and co‐electrophoresed with the two mouse myelin basic proteins could be detected. In control experiments with liver poly A(+) mRNA, which stimulated the translational system to a greater extent than brain mRNA, no such polypeptides could be detected. In this system the ratio of synthesis of small myelin basic protein to large myelin basic protein was found to be about 4.0, which correlates well with that found in vivo.Matthees J. and Campagnoni A. T. Cell‐free synthesis of the myelin basic proteins in a wheat germ system programmed with brain messenger RNA. J. Neurochem.35, 867–872 (1980).
Thymidine Transport in the Central Nervous System
Authors: Spector Reynold
Published: October 2006

The mechanisms by which thymidine enters and leaves brain, choroid plexus, and CSF were investigated by injecting [3H]thymidine intravenously and intraventricularly. [3H]thymidine, with and without unlabeled thymidine, was infused at a constant rate into conscious adult rabbits. At 150 min, [3H]thymidine readily entered CSF, choroid plexus, and brain. In brain, approximately 45% of the nonvolatile radioactivity was [3H]thymidine phosphates. The addition of 0.21 mmol/kg unlabeled thymidine to the infusion syringe decreased the phosphorylation of [3H]thymidine in brain by approximately 85%; the addition of 2.1 mmol/kg of unlabeled thymidine to the infusion syringe decreased the relative entry of [3H]thymidine into CSF and brain by 40 and 78%, respectively. Two h after intraventricular injection of [3H]thymidine, [3H]thymidine was rapidly cleared from CSF, in part, to brain, where approximately 40% of the [3H]thymidine was converted to [3H]thymidine phosphates. The intraventricular injection of unlabeled thymidine (21 μmol) with the [3H]thymidine abolished the phosphorylation of [3H]thymidine in brain and significantly decreased the clearance of [3H]thymidine from the CSF. Rabbit brain slices accumulated [3H]thymidine by an energy‐dependent, saturable high‐affinity system that depended, in part, on intracellular phosphorylation of the [3H]thymidine. These results were interpreted as showing that the entry of thymidine from blood into CSF and presumably the extracellular space of brain and then into brain cells involves one or more saturable transport and/or metabolic steps.
3H]Spiroperidol Binding in Rat Striatum: Two High‐Affinity Sites of Differing Selectivities
Authors: Andorn Anne C. , Maguire Michael E.
Published: October 2006

[3H]Spiroperidol binding to homogenates of rat striatum is saturable and shows either monophasic or biphasic saturation isotherms under specified conditions. In poorly washed membrane fragment preparations, saturation isotherms of [3H]spiroperidol binding are monophasic, revealing an apparently homogeneous set of sites with Kp 0.6 ± 0.3 nm and density 440 ± 80 fmol/mg protein. However, equilibrium displacement studies of [3H]spiroperidol binding at this site indicate an a‐adrenergic component in addition to the previously described dopaminergic component. In thoroughly washed membrane fragment preparations, saturation isotherms are clearly biphasic, showing an additional high‐affinity site with an approximate KD of 24 ±10 pm and an approximate density of 110 ± 20 fmol/mg protein at a protein concentration of 2.0 mg/ml. Selectivity at this site appears classically dopaminergic, suggesting that the lower affinity site is the primary source of the α‐adrenergic component of spiroperidol binding.
Hypotaurine Uptake by Brain Slices from Adult and 8‐Day‐Old Mice
Authors: Oja S. S. , Kontro P.
Published: October 2006

Abstract: Uptake of [35S]hypotaurine by brain slices prepared from adult and 8‐day‐old mice was studied at varying temperatures, under O2 and N2 atmospheres, and in the presence of metabolic inhibitors and varying concentrations of hypotaurine in the incubation medium. The tissue/medium concentration gradients generated were exceptionally high for an amino acid. Hypotaurine uptake was energy‐ and temperature‐dependent, more strictly in adult mice. Uptake was saturable, containing a high‐affinity and a low‐affinity component. The estimated transport constants for the high‐affinity uptake of hypotaurine (8‐day‐old mice, 17.2 μ mol/liter; adults, 35.3 μ mol/liter) were of the same order of magnitude as the reported transport constants of putative amino acid transmitters, but the total transport capacity appears to be greatest for hypotaurine.
Effect of Cerebellar Lesions on Monoamine Levels in Various Brain Areas of the Cat
Authors: Cano J. , Garcia‐Uria J. , Machado A. , Reinoso‐Suarez F.
Published: October 2006

Abstract: Modifications in the content of monoamines after different lesions of the cerebellar cortex were investigated in eight prosencephalic structures of cat's brain. Apart from other minor changes, lesions of the posterior vermis induced significant changes in the thalamus (decrease of DA and increase of 5‐HT). Lesions of the cortex of a cerebellar hemisphere, on the other hand, produced an increase of 5‐HT in the caudate nucleus and an increase of DA in the hippocampus in addition to a generalized increase of 5‐HT in all the prosencephalic structures studied. These findings are discussed in relation to the anatomical connections of the lesioned areas and their expected role in the sleep‐wakefulness cycle.
The Effect of γ‐Aminobutyric Acid on Substrate‐Level Phosphorylation in Brain Mitochondria
Authors: Rodichok Lawrence D. , Albers R. Wayne
Published: October 2006

Abstract: A consequence of the metabolism of γ‐aminobutyric acid (GABA) via the “GABA shunt” should be a decreased rate of substrate‐level phos‐ phorylation of GDP to GTP. 32P1 labeling of nucleotides was, therefore, studied in uncoupled brain mitochondria with α‐ketoglutarate or a‐ketoglutarate + GABA as substrates. The addition of an equimolar amount of GABA resulted in an approximately 50% reduction of the final specific activity of all mitochondrial nucleotides. This effect was completely reversed by aminooxyacetic acid. GABA did not affect the time course of nucleotide labeling. Although delineation of the mechanism involved requires further study, these preliminary results suggest an important modulatory role of GABA in the intermediary metabolism of brain mitochondria.
Isolation of 2′,3′‐Cyclic Nucleotide 3′‐Phosphodiesterase from Human Brain
Authors: Sprinkle Terry Joe , Grimes Marilyn J. , Eller A. Gary
Published: October 2006

Abstract: The enzyme 2′,3′‐cyclic nucleotide 3′‐phosphodiesterase (EC 3.1.4.37) has been isolated from an acetone powder of human subcortical white matter. The yield was about 11 mg from 28 g of powder and a specific activity of 213 unitdmg protein was obtained using 2′,3′‐cyclic CMP as the substrate. A major protein band of molecular weight approx. 96,000 was found by gel electrophoresis under nonreducing conditions. However, two distinct protein bands of molecular weight 46,000 ± 1400 and 48,000 ± 1400 were observed when the protein sample was reduced with 10 mM‐dithiothreitol and subjected to electrophoresis in more restrictive 12‐15% polyacrylamide‐SDS gels. This molecular weight is lower than that previously reported for the bovine enzyme. Antibodies against the purified human enzyme have been raised in New Zealand white rabbits.
Regulation of Muscarinic Acetylcholine Receptor Concentration in Cloned Neuroblastoma Cells
Authors: Shifrin Gary S. , Klein William L.
Published: October 2006

Abstract: The concentration of muscarinic acetylcholine receptors in the neuroblastoma cell line NIE‐115 is regulated by receptor activation. Muscarinic agonists cause a time and dose‐dependent loss of [3H]quinuclidinyl benzilate binding sites from cultured cells. Muscarinic antagonists have no effect on receptor concentration and block agonist‐induced regulation. The maximum decrease in steady state receptor levels is 80% and occurs within 9 h. The altered steady state concentration persists as long as agonist remains present. Upon withdrawal of agonist, the concentration of receptors returns to control levels. This increase requires protein synthesis. Kinetically, the increase in receptors following withdrawal of agonist is slower than the decrease caused by addition of agonist, suggesting that bursts of receptor activation could lower receptor levels. In harmony with this prediction, cycles in which receptors are active for 15 min and then inactive for 15 min cause a 50% decrease in receptor concentration in a 6‐h period.
Concanavalin A‐Sepharose Affinity Chromatography of Axon Plasma Membrane Proteins. Heterogeneity of Cholinergic Binding Sites
Authors: Marquis Judith K. , Hilt Dana C. , Mautner Henry G.
Published: October 2006

Abstract: Lysolecithin‐solubilized proteins from axon plasma membranes of lobster walking leg nerve bundles were chromatographed on concanavalin A (Con A)‐sepharose. Bound glycoproteins were eluted with α‐methyl‐D‐ mannoside. Near quantitative recovery of total protein was observed, 20–30% of the total protein being eluted in the Con A‐binding glycoprotein fraction. A 5‐fold enrichment of acetylcholinesterase (AChE) activity was achieved, demonstrating the glycoprotein nature of the axonal enzyme. The chromatographed fractions were characterized for binding of [3H]quinuclidinyl benzilate (QNB), [3nicotine (Nic), and [1251]α‐bung arotoxin (BgTx) in an attempt to distinguish possible “muscarinic” and “nicotinic” binding sites in axonal membranes. All of the high‐affinity “muscarinic” [3H]QNB binding activity appeared in the non‐Con A‐binding protein fractions, while binding of the two “nicotinic” ligands, [3Nic and 125I‐BgTx, was found in both the glycoprotein and non‐Con A‐binding protein fractions. BgTx interaction with the Con A‐binding glycoproteins could be blocked with dtubocurarine, but BgTx binding in the non‐Con A‐binding proteins was not inhibited by curare. The significance of multiple cholinergic binding sites in axonal membranes is discussed. These data suggest a closer similarity between the cholinergic ligand binding proteins of peripheral nerve membrane and ganglia than between the axonal cholinergic binding sites and the ACh receptor of the neuromuscular junction.
Changes in Glycoprotein Metabolism in the Cerebral Cortex Following First Exposure of Dark‐reared Rats to Light
Authors: Burgoyne Robert D. , Rose Steven P. R.
Published: October 2006

Abstract: The level of incorporation of [3H]fucose or [3H]lysine into subcel‐lular fractions of the visual and motor cortices of 50‐day‐old dark‐reared (D) and light‐exposed (L) rats was determined. No differences were found between D and L rats in the incorporation of either precursor into subcellular fractions of the motor cortex, or in any fraction of the visual cortex except the synaptic‐membrane fraction. After a 3‐h light exposure the incorporation of [3H]fucose into the visual cortex synaptic‐membrane fraction was elevated (L/D = 136%). Incorporation of [3H]lysine was elevated in the visual cortex synaptic‐membrane fraction of L compared to D rats after a 1‐h exposure (L/D = 118%). However, after a 3‐h exposure the incorporation was depressed in this fraction (L/D = 79%). No differences could be found in the levels of activity of fucosyl transferase following first exposure to light but dark‐rearing itself resulted in increased enzyme activity in the motor cortex compared to normal controls. First exposure of 20‐day‐old dark‐reared rats to light led to an increase in the incorporation of [3H]fucose into soluble glycoproteins of both the visual and motor cortex and into particulate glycoproteins of the visual cortex only. These results are in contrast with those found with 50‐day‐old animals and suggest that the effects of light‐exposure on [3H]fucose incorporation may be age‐dependent.
Neuronal Localization of Ca2+‐dependent Protein Phosphorylation in Brain
Authors: Sieghart Werner , Schulman Howard , Greengard Paul
Published: October 2006

Abstract: The cellular localization of two Ca2+‐dependent protein phosphorylation systems was investigated using the kainic acid lesioning technique for the selective destruction of neurons. In one of these systems, a crude synaptosomal (P2) fraction was preincubated with 32Pj for 30 min; the phosphorylation of several proteins was increased during a short subsequent incubation with veratridine plus Ca2+. In the second system, crude synaptosomal membranes isolated from the P2 fraction were incubated with [γ‐32P]ATP; in this system, the phosphorylation of several proteins was increased in the presence of a “calcium‐dependent regulator” plus Ca2+. Kainic acid lesioning greatly reduced the amount of Ca‐+‐dependent protein phosphorylation in both systems. The results indicate a predominantly neuronal localization for both Ca2+‐dependent protein phosphorylation systems.
Relationship Between the Actions of Calcium Ions, Opioids and Prostaglandin E1 on the Level of Cyclic AMP in Neuroblastoma × Glioma Hybrid Cells
Authors: Brandt M. , Buchen C. , Hamprecht B.
Published: October 2006

Abstract: Neuroblastoma × glioma hybrid cells increase their intracellular concentration of cyclic AMP in response to prostaglandin E1 (PGE1). This effect is inhibited by opioids. The response to PGE1 is positively correlated with the concentration of Ca2+ in the incubation medium. The Ca2+ antagonists Co2+ and La3+, the Ca2+ chelator EGTA and a blocker of Ca2+ influx into cells, Segontin, inhibit the response to PGE1. At low external concentrations of Ca2+ the response to PGE1 is enhanced by the Ca2+ ionophore A23187. The effects of A23187 and Segontin point to a cytosolic site of Ca2+ action. Lack of Ca2+ reduces the level of cyclic AMP even in the absence of PGE1 and the presence of an inhibitor of cyclic AMP phosphodiesterase. Ca2+ is required even for an increase in the level of cyclic AMP in cells pretreated with cholera toxin. The increases in level of cyclic AMP evoked by PGE, in a neuroblastoma and by PGE1 or noradrenaline in a glioma cell line do not depend on Ca2+. The response of the hybrid cells to the opioid leucine‐enkephalin appears not to rely on the presence of Ca2+. Even changing the intracellular concentration of Ca2+ by the ionophore A23187 does not alter the effect of the opioid. The analogy between opioids and lack of Ca2+ in the short‐term (minutes) experiments mentioned holds also for long‐term (hours) experiments. Cells chronically exposed to opioids or to low concentrations of Ca2+ display an enhanced maximal response to PGE1.
Neurochemical Characteristics of Myelin‐like Structure in the Chick Retina
Authors: Kohsaka Shin‐ichi , Takamatsu Ken , Nishimura Yozo , Mikoshiba Katsuhiko , Tsukada Yasuzo
Published: October 2006

Abstract: Certain characteristics of myelin‐like structures in the chick retina were examined morphologically and biochemically. Developmental changes of 2′, 3′‐cyclic nucleotide 3′‐phosphohydrolase (CNPase) in the chick retina and optic nerve were examined. The measurable activity in the retina was first detected at 16 days of incubation and thereafter, it increased rapidly until 4 weeks post‐hatching. By contrast, CNPase activity in the optic nerve reached the maximum level at 4 days post‐hatching and maintained a constant level thereafter. The purifed myelin fraction from the chick retina showed higher activity of CNPase, whereas its activity in the retinal homogenate was very low. Hence, it was considered that the myelin fraction from the chick retina is similar to that of CNS myelin with respect to CNPase. Protein profiles of the purified myelin fractions isolated from the chick optic tectum, optic nerve, retina and sciatic nerve were analysed by SDS‐polyacrylamide gel elec‐trophoresis. Myelin fractions from the chick optic tectum and optic nerve contained basic protein (BP) and Folch‐Lees proteolipid protein (PLP). Myelin fraction from the chick sciatic nerve contained BP, P2 and two glycoproteins (PO and 23K). In contrast, retinal myelin fraction contained only BP. PLP, PO, 23K and P2 proteins were definitely undetectable. Electron micrographs revealed that some axons in the optic nerve fiber layer of the chick retina were wrapped by a spiral‐structured myelin‐like sheath, which showed some differences from those of CNS and PNS myelin sheaths. It was suggested that the origin of the myelin‐like structure in the chick retina is other than from oligodendroglia or Schwann cells.