bluuebunny/crossref_metadata_2025 huggingface dataset:
THE CELL‐FREE SYNTHESIS OF ALKALOID‐BINDING PROTEINS IN IMMATURE RAT BRAIN1
Authors: Raeburn S. , Twomey S. L. , Baxter C. F.
Published: October 2006

Abstract— cell‐free amino acid incorporating system from immature rat brain, consisting of ribosomal and soluble fractions, has been investigated for its capacity to incorporate [14C]amino acids into specific soluble proteins that interact with vinblastine sulfate and colchicine. The soluble 14C‐labeled proteins formed in the cell‐free system during incubation were compared with similar soluble proteins from immature rat brain which had been labeled in vivo by the incorporation of 14C‐labeled amino acids. Criteria for the formation of vinblastine‐binding, 14C‐labeled proteins were: (1) aggregation of 14C‐labeled soluble protein by one mm‐vinblastine sulfate and (2) immunoprecipitation of 14C‐labeled soluble protein by an antiserum against vinblastine sulfate‐precipitable material. Criteria for the formation of [3H]colchicine‐binding, 14C‐labeled protein were based upon: (1) co‐precipitation of the 3H‐and 14C‐labeled materials by vinblastine sulfate and (2) the coincidence of 3H‐ and 14C‐labeled elution peaks from columns of Sephadex G‐200, DEAE‐Sephadex A‐50 and isoelectric focusing. Both in the in vitro and in the in vivo system, 14C‐labeled amino acids were incorporated into soluble proteins of the post‐microsomal supernatant fraction. Proteins labeled with 14C‐labeled amino acids in vitro and in vivo yielded comparable and qualitatively identical results by the criteria tested, including the formation of immunoprecipitates. In the in vitro system, 14C‐labeled amino acids were incorporated into protein with a molecular weight of approx 120,000, an isoelectric point of 5.3 and with a chromatographic mobility on Sephadex G‐200 which is identical to [3H]colchicine‐binding protein. The above experimental results are presumptive evidence for the synthesis of vinblastine‐binding and colchicine‐binding proteins in the in vitro cell‐free system.
BRAIN CARBONIC ANHYDRASE: ACTIVITY IN ISOLATED MYELIN AND THE EFFECT OF HEXACHLOROPHENE
Authors: Cammer W. , Fredman T. , ROSE A. L. , Norton W. T.
Published: October 2006

Abstract— Animals receiving hexachlorophene (HCP) in their diet develop cerebral edema with vacuolation of the myelin sheath. When carbonic anhydrase activities were measured in homogenates of brains from HCP‐fed and control rats, the HCP‐fed rats showed small decreases in the enzyme activity, but these changes were not statistically significant. HCP did inhibit the enzyme in vitro but at higher concentrations (10−5‐10−4m) than have been reported for HCP levels in brains of experimental animals. Carbonic anhydrase activity was present in myelin preparations obtained by gradient centrifugation and osmotic shock or by subcellular fractionation. When the latter procedure was used, myelin carbonic anhydrase had a specific activity which was higher than that of the mitochondrial fraction. The myelin enzyme was inhibited by 10−910−8m‐acetazolamide and, like the homogenates and the commercial enzyme, was inhibited by HCP. The mechanism for HCP toxicity remains unknown, but this study does suggest that carbonic anhydrase is an intrinsic component of the myelin sheath.
THE EFFECTS OF OUABAIN AND THE ACTIVATION OF NEURAL MEMBRANE ATPase BY BIOGENIC AMINES
Authors: Logan J. G. , O'Donovan D. J.
Published: October 2006

Abstract— The effects of 10−5m‐noradrenaline (NA), 5‐hydroxytryptamine (5‐HT) and dopamine (DA) on the activities of Na+‐K+ ATPase (EC 3.6.1.3) were studied in synaptic membranes from 6 regions of the rabbit brain. NA and 5‐HT stimulated the synaptic membrane Na+‐K+ ATPase from the cerebrum, but none of the amines influenced the activity of this enzyme in the other brain regions. The Na+‐K+ ATPase activity of the cerebral synaptic membrane isolated at the 0.8/0.9 m & 0.9/1.0 m interphase of a sucrose density gradient was increased two‐fold by 10−5m‐NA and 5‐HT. The Na+‐K + ATPase recovered at the 1.0/1.2 m interphase was not influenced by NA, DA or 5‐HT. NA, DA and 5‐HT did not activate the Mg ATPase of synaptic membranes from any of the 6 brain regions or whole brain synaptic vesicles. The cortex synaptic membrane (Na+‐K+) ATPase is postulated to have a direct role in the uptake of the biogenic amines. An indirect role is proposed for this enzyme in amine uptake into brain stem.
GAMMA‐HYDROXYBUTYRATE DEGRADATION IN THE BRAIN IN VIVO: NEGLIGIBLE DIRECT CONVERSION TO GABA
Authors: Möhler H. , Patel A. J. , Balázs R.
Published: October 2006

Abstract— The metabolism of γ‐hydroxybutyrate (GHB) was studied by following the fate of [1‐14C]GHB in mouse brain after an intravenous injection. Cerebral uptake of GHB was rapid and this substance disappeared from brain tissue with a half‐life of approx 5 min. Degradation of [1‐14C]GHB took place in the brain since 14C was incorporated in amino acids associated with the tricarboxylic acid cycle: the labelling pattern was consistent with the oxidation of GHB via succinate through the cycle, rather than with β‐oxidation of GHB. Conversion of [14C]GHB into [14C]GABA prior to oxidation was negligible, thus it is unlikely that the pharmacological action of GHB would be mediated through GABA formation. [14C]GHB oxidation also elicited the signs of metabolic compartmentation of the tricarboxylic acid cycle in the brain (glutamine/glutamate specific radioactivity ratio was about 4).
ATP HYDROLASE ACTIVITY ASSOCIATED WITH MICROTUBULES REASSEMBLED FROM BOVINE SPLENIC NERVE–A CAUTIONARY TALE
Authors: Banks P.
Published: October 2006

Abstract– ATP‐hydrolase activity is present in microtubules prepared from extracts of bovine splenic nerve.

The reassembled microtubules contain several proteins in addition to tubulin, including two which appear to be similar to the ‘dynein‐like’ polypeptides found in preparations of brain microtubules by other authors.

The sensitivity of the ATP‐hydrolase activity to Na+ and K+ ions and to ouabain suggests that it can be ascribed to a membrane‐bound sodium transport ATP‐hydrolase rather than to ‘dynein‐like’ polypeptides. This view is supported by the presence of phospholipids in the microtubule preparations even though membrane fragments could not be demonstrated by electron microscopy.
THE METABOLIC TURNOVER OF MOLECULAR SPECIES OF PHOSPHATIDYLINOSITOL and ITS PRECURSOR PHOSPHATIDIC ACID IN GUINEA‐PIG CEREBRAL HEMISPHERES
Authors: Luthra M. G. , Sheltawy A.
Published: October 2006

Abstract– The molecular species composition of phosphatidylinositol from guinea‐pig cerebral hemispheres was studied and found similar to that of phosphatidylinositol from ox cerebral hemispheres. In both cases the tetraenoic species was predominant. Phosphatidic acid from guinea‐pig cerebral hemispheres contained two major molecular species; the monoenoic and hexaenoic (33.4 and 24 mol/100 mol respectively).

In order to study the metabolism of molecular species of phosphatidic acid and phosphatidylinositol in the cerebral hemispheres, guinea‐pigs were injected intracisternally with 32Pi and [U‐14C]glucose. After 5 min of isotopic exchange, the specific radioactivity of 32P in phosphatidylinositol was nearly equal to that in phosphatidic acid, whereas specific radioactivity of 14C in the glycerol was 1.4 times and in the fatty acids nearly 0.5 times that in the phosphatidic acid respectively, indicating metabolic heterogeneity of both phospholipids.

The glycerol specific radioactivity was different in all the molecular species of phosphatidic acid being greatest in the monoenoic and least in the tetranenoic species. When the molecular species were arranged in this way, the order was representative of their relative rates of synthesis by acylation of glycerol‐3‐phosphate. An almost opposite order was obtained when the molecular species were arranged according to their phosphate/glycerol radioactivity ratios, indicating the relative contribution of the diacylglycerol kinase pathway to their formation.

When the specific radioactivity values and ratios of phosphatidylinositol were similarly considered, the orders of the molecular species were, on the whole, similar to that of phosphatidic acid. This indicated that synthesis de novo (Paulus & Kennedy, 1960) was operative in the formation of most of its molecular species, but due to other considerations it was concluded that part of the tetraenoic, and probably the whole of saturated phosphatidylinositol may be formed by transacylation reactions.

The results are discussed in terms of the experimental limitations of previous and present techniques for the analysis of phospholipid molecular species.
PYRIMIDINE BIOSYNTHESIS AND ITS REGULATION IN THE DEVELOPING RAT BRAIN1
Authors: Tremblay G. C. , Jimenez U. , Crandall D. E.
Published: October 2006

—Measurements of the incorporation of [14C]NaHCO3 into orotic acid, uridine nucleotides and RNA in tissue minces establish the occurrence of the complete orotate pathway for the de novo biosynthesis of pyrimidines in rat brain. Selective inhibition of the incorporation of various radiolabelled precursors into orotic acid by uridine demonstrates the operation of a feedback control mechanism in brain minces and indicates carbamoylphosphate synthetase to be the site of inhibition; purine nucleosides were similarly found to inhibit the de novo biosynthesis of pyrimidines.

The activity of the orotate pathway, as assessed by the rate of incorporation of [14C]NaHCO3 into orotic acid, was found to be very high in fetal brain and to decline rapidly with neurological development; the mature rat brain exhibits less than 1% of the activity of the fetal brain at 18 days of gestation. Comparative studies on the ability of minces of the brain and several extraneural tissues to utilize [14C]NaHCO3 and [14C]aspartate as precursors of orotic acid lead us to speculate that variations in the ability of tissues to synthesize orotic acid de novo are determined by similar variations in their ability to synthesize carbamoylphosphate.
EFFECTS OF TRYPTOPHAN ON SEROTONIN IN NERVE ENDINGS1
Authors: McBride W. J. , Hyde T. P. , Smith, J. E. , Lane J. D. , Aprison M. H.
Published: October 2006

—Preparations of crude synaptosome fractions (P2) from the telencephalon and from the diencephalon plus optic lobes of the pigeon and from the telencephalon of the rat were used to study the effects of l‐tryptophan on (a) the levels of serotonin (5‐HT), norepinephrinc (NE) and dopamine in nerve endings and (b) the release of radioactive 5‐HT, NE and dopamine from nerve endings. The level of 5‐HT was significantly higher (P < 0–05) in the P2 fraction isolated from the telencephalon of pigeons given intramuscular injections of 300mg/kg of l‐tryptophan in comparison to control values (1.11 ± 0.09 vs 0.74 ± 0.13 nmol/g original tissue wt). A smaller but not statistically significant increase in 5‐HT was noted in the P2 fractions isolated from the diencephalon plus optic lobes of pigeons given injections of l‐tryptophan. In vitro studies using preparations of synaptosomes (from both pigeon and rat) labelled with [3H]5–HT demonstrated that 1.0 mm‐l‐tryptophan caused a 30% increase (P < 0.05) in the release of [3H]5‐HT over control values. This effect by l‐tryptophan was blocked when a decarboxylase inhibitor was added to the medium. Tryptophan had no effect on the levels of NE or dopamine in these nerve endings nor did it have any effect on the release of these two amines from these preparations of synaptosomes. The results are discussed in terms of the role of serotonin in producing depression in pigeons working on a certain learned behavioural task.
ORNITHINE AND GLUTAMIC ACID DECARBOXYLASE ACTIVITIES IN THE DEVELOPING CHICK RETINA
Authors: De Mello F. G. , Bachrach U. , Nirenberg M.
Published: October 2006

Abstract— γ‐Aminobutyric acid was found in 6‐day‐old chick embryo retina; approx 20% was formed from putrescine and the remainder from glutamic acid. However, at the 18th embryonic day only 1% of the γ‐aminobutyric acid synthesized in retina was derived from putrescine. These results show that γ‐aminobutyric acid is synthesized in chick embryo retina prior to synaptogenesis, and that γ‐aminobutyric acid is synthesized via two pathways. The first pathway involves the conversion of putrescine to γ‐aminobutyric acid; the second path is dependent upon the conversion of glutamic acid to γ‐aminobutyric acid, catalyzed by glutamic acid decarboxylase.
OCTOPAMINE IN MAMMALIAN BRAIN: RAPID POST MORTEM INCREASE AND EFFECTS OF DRUGS
Authors: Harmar A. J. , Horn A. S.
Published: October 2006

Abstract—

A modified enzyme radiochemical assay for octopamine, based upon the N‐methylation of octopamine by the enzyme phenylethanolamine N‐methyl transferase (S‐Adenosyl‐1‐methionine: phenylcthanolamine N‐methyl transferase EC 2.1.28), has been developed. [3H]Methyl‐S‐adenosyl‐l‐ methionine was used as methyl donor, and the reaction products separated by thin‐layer chromatography prior to liquid scintillation counting. The method had a sensitivity of about 100 pg, and was suitable for the measurement of endogenous octopamine levels in mammalian brain. Although the method could be used for the determination of phenylethanolamine with similar sensitivity, concentrations of this amine in brain were too low for routine measurement. Octopamine levels in the brains of a number of mammalian species were determined using this procedure. Concentrations of the amine in mouse brain were lower in animals killed by rapid freczing than in animals killed by decapitation; a further increase in brain octopamine took place post‐mortem. Brain octopamine was increased following treatment with MAO inhibitors, p‐chlorophenylalanine, phenylalanine, tyrosine or phenylethylamine. The effects of tyrosine and phenylethylamine were greatly increased by pretreatment with a monoamine oxidase inhibitor. The antidepressants imipramine and iprindole gave rise to increased brain octopamine concentrations, possibly through an effect upon monoamine oxidase. Administration of chlordiazepoxide chlorpromazine, thyroxine, or reserpine had no effect upon brain octopamine.
A STUDY OF GLYCOLYTIC AND PENTOSE PHOSPHATE SHUNT ENZYMES IN RELATIONSHIP TO THE ALTERED PERMEABILITY IN THE BLOOD‐BRAIN BARRIER
Authors: Bennett M. J. , Ogilvy. K. M. , Blake G. M. , Lewtas N. , Timperley W. R.
Published: October 2006

The activities of a number of glycolytic and hexose‐monophosphate shunt cnzymes in 14 cases of intracranial pathology have been compared with specific uptake of the radioactive tracer 99m Tc‐pertechnetate. Strong positive correlations were observed betwcen isotope uptake and the activities of hexokinase and glucose‐6‐phosphate dehydrogenase. No close correlation was observed with other enzymes studied. Hexokinase is considered to be one of the major rate determining enzymes for the glycolytic catabolism of carbohydratcs and the subsequent production of nucleotidc triphophosphates which are vital in active transport mechanisms. This could indicate an important role for this enzyme in blood‐brain barrier kinetics.
BIOCHEMTCAL CHANGES IN THE BRAIN OF EXPERIMENTAL ANIMALS IN RESPONSE TO ELEVATED PLASMA HOMOCYSTINE AND METHIONINE
Authors: Tudball N. , Griffiths R.
Published: October 2006

The effects of high plasma concentrations of homocystine and methionine on the free amino acids of brain have been examined. Incorporation of the label from [35S]methionine into the free amino acid pools of rabbit brain was enhanced in response to high plasma homocystine or high plasma homocystine and mcthionine. Under comparable conditions a marked decrease in the incorporation of the label from [14C]glycine into the free pool was observed. The corresponding incorporation of 35S and 14C into brain proteins parallelled the results obtained with incorporation into the free pools of amino acids. Amino acid analyses of the free amino acid pools of rabbit brain revealed a general decrease in the concentration of amino acids in response to high plasma homocystine or high plasma homocystine and methionine. Inhibition of protein synthesis which occurs under the above experimental conditions is a general phenomenon. myelin and other brain fractions being equally affected. The decrease in concentration of brain amino acids also results in a diminution in concentration of the neurotransmitters GABA, dopamine and noradrenaline. The possible relationship of the observed changes to homocystinuria is discussed.
INFLUENCE OF NEONATAL HYPOTHYROIDISM UPON TRANSCRIPTION IN ISOLATED RAT BRAIN AND LIVER NUCLEI
Authors: Krawiec L. , Montalbano C. A. , Gömez C. J.
Published: October 2006

The optimum conditions for measuring the transcriptional activity of endogenous RNA polymerase I and I1 were established in low ionic strength incubation mixtures for liver and cerebrum cell nuclei. Both activities were estimated in the presence of Mg2+ for RNA polymerase 1 and Mn2+ for RNA polymerase I1 under conditions in which the nuclear morphology is prcscrvcd. The UMPIGMP incorporation ratio was used to determine the type of RNA synthesized and the results were confirmed employing specific inhibitors of RNA synthesis, such as a‐Amanitin and Actinomycin‐D. Neonatal thyroidectomy in the rat diminished the normal growth of body and liver more than the cerebral growth. The transcriptional capacity in the presence of Mg2+ in cerebral cell nuclei was not changed by the absence of thyroid function during postnatal development. Contrariwise, the activity the presence of Mn2+ was transicntly diminished between the 10th and 13th day of life. When the assays were performed at high ionic strength there were no differences between normal and hypothyroid animals. In liver, thyroid deficiency affected in an early and permanent way the transcription of an AU‐rich RNA. The endogenous activity measured at high ionic strength and that tested in the presence of Mg2+ were altered from the 20th day onward.
OCCURRENCE OF γ‐GLUTAMYLHISTIDINE IN BOVINE BRAIN
Authors: Kakimoto Y. , Konishi H.
Published: October 2006

γ‐Glutamylhistidine was purified from 7.5 kg of bovine brain. ldentification was based on the comparison with the synthetized compound. γ‐Glutamyllysine and γ‐glutamylarginine were not detected. The present study added another γ‐glutamyl amino acid 10 the list of the previously known 10 γ‐glutamyl compounds.
SDS GEL ELECTROPHORESIS OF RAPIDLY TRANSPORTED PROTEINS IN GARFISH OLFACTORY NERVE
Authors: Cancalon P. , Elam J. S. , Beidler L. M.
Published: October 2006

Abstract— Proteins undergoing rapid axonal transport in the garfish olfactory nerve were examined by sodium dodecyl sulphate gel electrophoresis. The distribution of polypeptides and the extent of their labeling by transported molecules was determined in several nerve subfractions including: total particulate, total membrane, mitochondrial and two membrane subfractions rich in axolemma. The polypeptide composition of the various fractions was found to be relatively similar, with each showing a major protein with an estimated MW of 58,000. Specific differences in the concentrations of certain proteins were noted between fractions, including differences between the lower and higher density axolemma rich subfractions. Axonally transported radioactivity was predominantly localized among high molecular weight proteins, with all fractions, except mitochondrial pellet, displaying a major peak of radioactivity centered at 126,000‐MW. Several major proteins including the 58,000‐MW band were labeled by rapid transport to a much smaller extent. Certain labeled peaks were found to be concentrated in individual fractions, particularly a polypeptide (MW 35,000) more predominantly found in the lower density axolemma rich fraction.

Systemic labeling of the nerve is found to give a general distribution of radioactivity on gels, which is clearly different from the pattern obtained after axonal transport labeling.
STUDIES ON THE BIOSYNTHESIS OF GLYCO‐SPHINGOLIPIDS IN CULTURED MOUSE NEUROBLASTOMA CELLS: CHARACTERIZATION AND ACCEPTOR SPECIFICITIES OF N‐ACETYLNEURAMINYL‐ AND N‐ACETYLGALACTOSAMINYLTRANSFERASES
Authors: Kemp S. F. , Stoolmiller A. C.
Published: October 2006

Abstract— The pathway of biosynthesis of N‐acetylgalactosamine‐containing gangliosides in mouse neuroblastoma has been studied using NB41A cells grown in monolayer tissue culture. Cell‐free enzyme preparations catalyzed the transfer of NeuNAc from CMP‐NeuNAc to lactosylceramide (GL‐2a), to form GM3. Asialo‐GM2 was neither an acceptor nor a competitive inhibitor of the sialyltransferase (CMP‐NeuNAc: GL‐2a N‐acetylneuraminyltransferase, EC 2.4.99.‐) under a variety of experimental conditions. Enzyme preparations also contained an N‐acetylgalactosaminyltransferase (UDP‐GalNAc. GM3N‐acetylgalactosaminyltransferase, EC 2.4.1.‐) which catalyzed the conversion of GM3 to GM2. No significant transfer of N‐acetylgalactosamine to GL‐2a could be demonstrated. The results of the glycosyltransferase assays support the concept that the first NeuNAc of brain gangliosides is introduced into GL‐2a. The present data suggests that the occurrence of asialo‐GM2 in NB41A cells under some culture conditions is a consequence of the catabolism of higher gangliosides.
A RAPID ASSAY FOR 2′,3′‐CYCLIC NUCLEOTIDE 3′‐PHOSPHORYDROLASE1
Authors: Sims N. R. , Carnegil P. R.
Published: October 2006

Abstract— Separation of 2′‐adenosine monophosphate from 2′,3′‐cyclic adenosine monophosphate by coprecipitation with a number of salt mixtures was examined and found to be most successful with Na2CO3/CdCl2 and Na2CO3/ZnSO4. A simple and rapid assay for 2′,3′‐cyclic nucleotide 3′‐phosphohydrolase using coprecipitation with Na2CO3/CdCl2 is described.
MYELIN BASIC PROTEIN PHOSPHATASE ACTIVITY IN RAT BRAIN
Authors: McNamara J. O. , Appel S. H.
Published: October 2006

Abstract— Previous work from this and other laboratories has demonstrated phosphorylation of myelin BP in vivo and in vitro. The rapid turnover of BP phosphate has suggested the presence of a phosphatase. The present studies have identified two BP phosphatases. One is present in the cytosol of rat brain homogenate. It has the highest specific activity (37 pmol/min/mg) and total activity of BP phosphatase present in any subcellular fraction. The partially purified cytosol enzyme can readily dephosphorylate soluble 32P‐labelled BP but is only half as effective in dephosphorylating membrane‐bound BP. Conversely, the phosphatase which remains associated with highly purified myelin is 2.3 times as effective on BP in the membrane (7.2 pmol/min/mg) as on soluble BP (3.2 pmol/min/mg). The myelin phosphatase is tightly bound to the membrane and cannot be removed with concentrated salt solutions. During development the specific activity of the cytosol phosphatase remains constant. The specific activity of the myelin phosphatase, however, is twice as high during the period of maximum myelin formation (6.8 pmol/min/mg at 18 days) as it is in adult myelin (3.2 pmol/min/mg at 12 weeks).

In order to compare enzyme effectiveness under the various conditions employed in these studies, we have assumed that both soluble and particulate substrates are phosphorylated at equivalent sites on the polypeptide. We have further assumed that soluble and/or particulate substrates are dephosphorylated at equivalent sites on the polypeptide chain and that the various particulate and soluble enzymes have comparable access to the substrate. Within the limitations of these assumptions, our data suggest myelin phosphatase may play a significant role in phosphate turnover of BP.
INCREASE IN THE APPARENT AChE ACTIVITY IN THE MOUSE DIAPHRAGM INDUCED BY PROTEOLYTIC TREATMENT
Authors: Sketeij J. , Brzin M.
Published: October 2006

Abstract— Isolated endplate regions from the mouse diaphragm were treated with different agents before or after homogenization in order to solubilize junctional AChE and study the effect of solubilization on its apparent activity. Total AChE activity (solubilized + nonsolubilized) of samples treated with collagenase or papain before homogenization was nearly twice as high as in control samples. If collagenase was added after homogenization no increase in apparent activity was observed although in both cases about 70–80% of AChE activity was solubilized. The access of ACh to the membrane‐bound enzyme is probably not a limiting factor in the AChE assay as is the case in the electric organ homogenates. Both 1 m‐NaCl and Triton X‐100 were quite ineffective as solubilizers when applied before homogenization and had an insignificant effect on the apparent AChE activity.

The increase in apparent AChE activity cannot be explained either by a de novo synthesis or by the change in kinetic properties of different species of AChE, or by the release of AChE possibly sequestrated in the membrane vesicles. The possibility is discussed that a part of junctional AChE is inactivated at the beginning of homogenization while it can be preserved by previous solubilization, or that proteolytic treatment may activate some ‘silent’ AChE sites in motor endplates.

However, the mere fact that the difference does exist suggests that all AChE activity present in intact motor endplates may not be measurable after homogenization.
DOPAMINERGIC PROPERTIES OF A SOMATIC CELL HYBRID LINE OF MOUSE NEUROBLASTOMA X SYMPATHETIC GANGLION CELLS
Authors: Greene Lloyd A. , Rein Glen
Published: October 2006

Abstract— Studies were made on the regulation of dopamine metabolism in a cell line derived by hydridization of a non‐tyrosine‐hydroxylase‐containing line of murine neuroblastoma cells with a neur‐onally‐enriched population of murine embryonic sympathetic ganglion cells. Hybrid subclones with tyrosine hydroxylase activity were selected by exposure to tyrosine‐free medium. The cells also exhibited DOPA decarboxylase activity and the subclone (named T28) with the highest specific activities of both enzymes was further characterized. The hybrid T28 line did not contain dopamine‐β‐hydroxylase activity. The specific activity of tyrosine hydroxylase as well as of DOPA decarboxylase increased significantly in T28 cultures when the cells entered the stationary phase of growth. Both of these enzymes were also induced after several days of exposure to 1 mm‐dibutyryl cyclic AMP in culture medium containing either 5% or 0.8% serum. However, maintenance in medium containing 0.8% serum alone, which inhibited cell multiplication, did not induce either enzyme. The dopamine content of T28 cells was also regulated as a function of cell density. High density (stationary phase) cultures of T28 cells contained about 300 pmol dopamine per mg protein and at least half of this endogenous amine appeared to he stored in vesicles or granules (as judged by depletion with reserpine or α‐methyl‐m‐tyramine). The T28 and other neuronal hybrid lines appear to be useful model systems for neuro‐chemical studies.
THE EFFECTS OF CARBAMYLCHOLINE ON INCORPORATION IN VIVO OF [1‐14C]ARACHIDONIC ACID INTO GLYCEROLIPIDS OF MOUSE BRAIN
Authors: Lee Su Kwei , Sun Grace Y.
Published: October 2006

Abstract— The effects of carbamylcholine on incorporation of [1‐14C]arachidonate into the glycerolipids in mouse brain synaptosome‐rich and microsomal fractions were examined at 1, 3 and 10 min after intracerebral injection of the labeled precursor. When carbamylcholine was included with the labeled arachidonate, there was a decrease in the proportion of labeled fatty acid incorporated into the phospholipids. Among the phospholipids in the synaptosome‐rich fraction, a decrease in incorporation of radioactivity into diacyl‐glycerophosphoinositols and diacyl‐glycerophosphocholines was observed at 1 and 3 min after injection. A decrease in labeling of diacyl‐glycerophosphoethanolamines and diacyl‐glycerophosphocholines in the microsomal fraction was observed at 3 and 10 min after injection. The decrease in phospholipid labeling was marked by an increase in labeling of diacylglycerols which was observed initially in the synaptosome‐rich fraction, but also in the microsomal fraction at later time periods. Other lipid changes included an increase in triacylglycerol labeling which was found in the synaptosome‐rich fraction and an increase in phosphatidic acid labeling which was found in the microsomal fraction. Results of the in vivo study have demonstrated changes in brain lipid metabolism during carbamylcholine stimulation. Furthermore, these changes appear to be initiated mainly in the synaptosome‐rich fraction.
REGULATION OF THE LEVEL OF ENDOGENOUS PHOSPHORYLATION OF SPECIFIC BRAIN PROTEINS BY DIPHENYLHYDANTOIN1
Authors: Delorenzo R. J. , Emple G. P. , Glaser G. H.
Published: October 2006

Abstract—

Diphenylhydantoin (DPH) in therapeutic concentrations caused a decrease in the net level of endogenous phosphorylation of two specific proteins from rat brain cerebri, while not significantly affecting the phosphorylation of other protein substrates. The apparent molecular weights of the DPH‐ specific substrate proteins were 60‐63,000 and 49‐52,000, and were designated proteins DPH‐L and DPH‐M, respectively. DPH decreased both the initial rate and the net level of [32P] phosphate incorporation from [γ‐32P] ATP into proteins DPH‐L and DPH‐M. The concentrations of DPH required to produce a half maximal decrease in the levels of phosphorylation of proteins DPH‐L and DPH‐M was 3 × 10‐4 and 8 × 10 ‐4 M, respectively. The effects of DPH on the incorporation of [32P] phosphate into these specific brain proteins were independent of the concentration of ATP over a wide range of ATP concentrations. The DPH‐specific proteins were demonstrated to be present in synaptosomal preparations. The results are compatible with the hypothesis that some of the stabilizing actions of DPH on neuronal tissue and seizure discharge may be mediated by the effect of this anticonvulsant on the phosphorylation of specific brain protein substrates.
NUCLEAR DNA‐DEPENDENT DNA POLYMERASES IN HUMAN BRAIN
Authors: Viola M. V. , Norton P. , Cole Margery L.
Published: October 2006

Abstract—

DNA‐dependent DNA polymerases were partially purified from nuclei of cells from the occipital lobe of human brain. The purification procedure included successive DEAE‐cellulose and phosphocellulose column chromatography, gel filtration and sucrose density gradient centrifugation steps. Four enzymes corresponding to DNA polymerases‐α, β, γ, and terminal deoxynucleotidyl transferase were found. Brain DNA polymerases could be differentiated from one another by size, template preferences and sensitivity to sulfhydryl blocking agents.
AXONAL TRANSPORT OF [35S] SULPHATE IN RETINAL GANGLION CELLS OF THE RABBIT
Authors: KARLSSON J. ‐O. , LINDE A.
Published: October 2006

Abstract—

A rapid axonal transport of 35SO4 in axons of retinal ganglion cells of the rabbit was demonstrated after intraocular administration of the isotope. Transported radioactivity accumulated in the nerve terminals up to 2 days following isotope injection. No evidence for a slow transport of labelled material was found. A considerable part of transported radioactivity had solubility characteristics of glycosaminoglycans, primarily chondroitin sulphate.
METHYL MERCURY INHIBITION OF SYNAPTOSOME AND BRAIN SLICE PROTEIN SYNTHESIS: IN VIVO AND IN VITRO STUDIES
Authors: VERITY M. A. , BROWN W. J. , CHEUNG M. , CZER G.
Published: October 2006

Abstract—

Subacute methyl mercury (MeHg) intoxication was induced in adult rats following the daily intragastric administration of 1 mg MeHg/100 g body weight. Decreased [14C]leucine incorporation into cerebral and cerebellar slice protein was found. Weight loss occurred during the latent and neurotoxic phases but pair feeding did not reveal a significant defect in amino acid incorporation into slice protein. There was no decline in synaptosome protein synthesis in vitro during the latent phase but a significant decline in cerebellar and cerebral synaptosome synthesis was found during the neurotoxic phase. MeHg in vitro inhibited cerebral slice and synaptosome protein synthesis at half maximal concentrations of 7.5 and 12.5 μM respectively. Inhibition of synthesis in synaptosomes was non‐competitive with K1 of 4 × 10−6M. MeHg had no effect on [14C]leucine or [14C]proline uptake into synaptosomes. There was no significant inhibition of synaptosome basal ATPase or Na + K ATPase at concentrations of MeHg (12 μM) giving half maximal inhibition of protein synthesis. No preferential inhibition of the chloramphenicol (55S) or cycloheximide sensitive components of synaptosome fraction protein synthesis was found, suggesting that the inhibition is common to both mitochondrial and extramitochondrial protein synthesizing systems. Addition of nucleotides and/or atractylate failed to influence protein synthesis and did not reverse the MeHg inhibition. Mannitol, as a replacement for the predominant cation species of the incubation medium, gave 40% inhibition of protein synthesis in the control but protected against further inhibition by MeHg.
CHARACTERISTICS OF D‐GLUCOSAMINE UPTAKE BY RAT BRAIN SYNAPTOSOMES
Authors: Tan C. H. , Peterson N. A. , Raghupathy E.
Published: October 2006

Abstract— The uptake of D‐glucosamine by rat brain synaptosomes is studied as a function of time, temperature and synaptosomal protein and substrate concentrations. The rate of D‐glucosamine uptake, after correcting for simple diffusion, obeys Michaelis‐Menten kinetics. The apparent kinetic constants for the uptake process are Km= 2.5 0.8 mm, Vmax= 3.7 ± 1.2 nmol/mg protein/min. D‐Glucose, D‐mannose, 2‐deoxy‐D‐glucose and 3‐0‐methyl‐o‐glucose are potent inhibitors of D‐glucosamine uptake. 2‐Deoxy‐D‐glucose and D‐glucosamine inhibit the uptake of one another in a simple competitive manner, indicating their sharing of a common transport system. Cytochalasin B, phloretin and phloridzin are powerful competitive inhibitors of D‐glucosamine uptake with apparent inhibitor constants (K1) of 7.0 × 10‐5, 2.3 × 10‐3 and 0.4 mM, respectively. The uptake is unaffected by Na+, Li+ and Mg2+, partially inhibited by NH4+, Mn2+ and Ca2+, and slightly stimulated by PO4‐ions. D‐Glucosamine uptake is also sensitive to inhibition by several sulfhydryl reagents, thus implying the involvement of sulfhydryl groups in the transport process. The apparent affinity constants for synaptosomal transport for both D‐glucosamine and 2‐deoxy‐D‐glucose are about 4 times greater in 7‐day‐old than in the adult rat brains.
HIGH AFFINITY UPTAKE SYSTEMS FOR TAURINE IN TISSUE SLICES AND SYNAPTOSOMAL FRACTIONS PREPARED FROM VARIOUS REGIONS OF THE RAT CENTRAL NERVOUS SYSTEM. CORRECTION OF TRANSPORT DATA BY DIFFERENT EXPERIMENTAL PROCEDURES
Authors: Lombardini J. B.
Published: October 2006

Abstract— —The uptake of taurine into tissue slices of specific regions of the rat central nervous system (CNS) was compared with the uptake of taurine into synaptosomal fractions prepared from the corresponding regions. Two different techniques for performing control experiments were also compared: procedure I, correction for the uptake of taurine obtained from duplicate incubations but at 2°c and procedure II, correction of taurine uptake into extracellular or extrasynaptosomal space measured by inulin uptake experiments plus correction for diffusion (non‐saturable) processes.

Kinetic analyses of the uptake data in tissue slices utilizing the procedure I correction technique indicate that six regions of the rat CNS (spinal cord, diencephalon, cortex, striatum, hippocampus, and midbrain) possess high affinity uptake systems (Km values approx 60 μM or less). The Km value for the cerebellum (105.4 ± 15.7 μM) is intermediate between a high and low affinity uptake system while the Km value for the pons‐medulla (210.0 12.4 μM) is considered to be low affinity. When procedure II techniques were utilized for correcting the uptake data all eight regions demonstrated high affinity uptake systems (11.8–73.2μM).

Synaptosomal fractions prepared from the spinal cord, pons‐medulla, diencephalon, and midbrain demonstrate high affinity uptake systems (procedure I) for taurine (10.3–47.2 μM) while the hippocampus, cortex, striatum, and cerebellum have intermediate (but still high affinity) values (59.4–96.4 μM). High affinity uptake systems (8.2–79.8 μM) were obtained for all eight regions of the rat CNS when procedure II was utilized for correction of the data.
DIFFERENTIATION IN BRAIN AND LIVER DOPA/SHTP DECARBOXYLASE ACTIVITY AFTER L‐DOPA ADMINISTRATION WITH OR WITHOUT PYRIDOXINE IN CATS1
Authors: Roberge A. G.
Published: October 2006

Abstract— Pyridoxine (50mg/kg, per os) given for 7 consecutive days did not modify the content of dopamine, noradrenaline, and serotonin in the neostriatum of the brain 3, 6 and 18 h after the last dose, but significantly increased DOPA/5HTP decarboxylase activity in both the neostriatum and liver. The administration of l‐DOPA and pyridoxine (100 and 50mg/kg, per os, respectively) together for 7 days increased DOPA/5HTP decarboxylase activity in the brain to the same extent as did l‐DOPA and pyridoxine given individually. Liver DOPA/5HTP decarboxylase activity remained normal when both drugs were administered together. However it decreased significantly after l‐DOPA administration for 7 days but not after pyridoxine treatment. In cats under treatment with l‐DOPA for 7 days, actinomycin D given for the final 3 days prevented the increased DOPA/5HTP decarboxylase activity induced by l‐DOPA in the neostriatum and mesencephalon but had no effect on the enzymatic activity in the liver. These findings indicate that differences exist between brain and liver DOPA/SHTP decarboxylase activity in uivo. In addition, denatured supernatant from livers of animals treated with l‐DOPA contained a dialysable compound which inhibits DOPA/SHTP decarboxylase activity in the supernatant from livers of untreated cats. In animals who received pyridoxine along with l‐DOPA, no such inhibitor was found. These results may explain the mechanism by which l‐DOPA exerts its beneficial effects and why pyridoxine administered with l‐DOPA reduces the therapeutic effectiveness of l‐DOPA in Parkinson's disease. These findings are consistent with the possibility that a tetrahydro‐isoquinoline derivative formed in vivo in the liver after l‐DOPA therapy for 7 days might affect DOPA/5HTP decarboxylase activity in the liver but not in brain. A tetrahydroisoquinoline derivative did not appear to be formed when l‐DOPA and pyridoxine were administrated together suggesting that pyridoxine protected the enzyme and favored a more rapid degradation of l‐DOPA peripherally with less l‐DOPA available for the CNS.
IN VITRO PROTEIN SYNTHESIS ON FREE POLYRIBOSOMES ISOLATED FROM THE DEVELOPING MOUSE BRAIN
Authors: Campagnoni A. T. , Harris J. R.
Published: October 2006

Abstract— Free polyribosomes were isolated at three ages (newborn, 2 weeks and 8 weeks) from the developing mouse brain. All three preparations were found to be highly polyribosomal in nature and were essentially identical with respect to their chemical composition and sedimentation properties. An estimate of the sedimentation coefficients of the first seven members of these polysome preparations yielded S°20,w values of 76, 114, 146, 174, 196, 217 and 236. All three preparations were found to be very active when employed in in vitro protein synthesizing systems. An age‐dependent response to the concentration of K+ was observed in the activities of the in vitro protein synthesizing systems. Optical K+ concentrations for the 0, 2 and 8 week old systems were 30, 50 and 65 mm, respectively. No such age dependence was observed when NH+4 was used as the sole monovalent cation, with all systems exhibiting maximal activity at 50mm‐NH+4. The highest in uitro activities were consistently observed (at all three ages) when NNH+4 was employed as the sole monovalent cation. Under optimal conditions, the newborn in vitro protein synthesizing system was observed to be approx 40% as active as either the 2 week or the 8 week systems which were equivalent in activity. The reduced activity of the newborn system appeared to be a function of both the polyribosomal and pH 5 enzyme preparations.
METABOLITES AND ENZYMES OF THE PENTOSE PHOSPHATE PATHWAY IN ISOLATED NERVE ENDINGS1
Authors: Kauffman F. C. , Harkonen M. H. A.
Published: October 2006

Abstract— The activities of each enzyme associated with the pentose phosphate pathway as well as the non‐enzymatic intermediates in this pathway were measured in synaptosomes isolated from rat cerebral cortex. The specific activities of transketolase (EC 2.2.1.1) and transaldolase (EC 2.2.1.2) were significantly lower in synaptosomes than cerebral cortex; however, the specific activities of glucose‐6‐phosphate dehydrogenase (EC 1.1.1.49), 6‐phosphogluconate dehydrogenase (EC 1.1.1.44), ribosephosphate isomerase (EC 5.3.1.6) and ribulosephosphate epimerase (EC 5.1.3.1.) were comparable in homogenates of synaptosomal fractions and cerebral cortex. Concentrations of most intermediates of the pentose pathway were also similar in extracts of synaptosomes and brain homogenates.

Six hours after treatment of rats with the nicotinamide analog, 6‐aminonicotinamide (6‐AN), 6‐phosphogluconate levels in synaptosomes were increased 5‐fold; however, glucose‐6‐phosphate levels remained unchanged. During a 30 min in uitro incubation 6‐phosphogluconate levels increased approx 2‐fold in synaptosomes obtained from 6‐AN treated rats but did not change in synaptosomes from untreated rats. During the same period glucose‐6‐phosphate levels decreased in synaptosomes from both control and 6‐AN treated rats. The conversion of both [1‐14C]glucose and [6‐14C]glucose to 14CO2 was depressed in synaptosomes from 6‐AN treated rats; however, the ratio of the two isotopes converted to 14CO2 was essentially the same. It is concluded that the pentose phosphate pathway is active in nerve endings both in vivo and in vitro.
ACETYLCHOLINE METABOLISM AND CHOLINE UPTAKE IN CORTICAL SLICES
Authors: Polak R. L. , Molenaar P. C. , van Gelder M.
Published: October 2006

Abstract— The uptake of [14C]choline was studied in cortical slices from rat brain after their incubation in a Krebs‐Henseleit medium containing either 4.7 mm‐KCl (low K), 25 mm‐KCl (high K) or 25 mm‐KCl without calcium (Ca free, high K). With 0.84 μm‐[14C]choline in the medium the uptake per gram of tissue was 0.62 nmol after incubation in low K medium, 1.13 nmol after incubation in high K medium and 0.78 nmol after incubation in a Ca free, high K medium. The differences caused by potassium were greater in fraction P2 than in fractions P1 and S2. With 17 and 50μm‐[14C]choline in the medium greater amounts of [14C]choline were taken up, but the effect of potassium on the uptake almost disappeared. The amount of radioactive material in fraction P2 followed Michaelis‐Menten kinetics with Km values of 2.1 and 2.3 μm after incubation in low and high K medium, respectively. Hemicholinium‐3 only slightly inhibited choline uptake from a medium with 0.84μm‐[14C]choline, but it abolished the extra‐uptake induced by high K medium. The radioactivity in the slices consisted mainly of unchanged choline and little ACh was formed after incubation in low K medium, but after incubation in high K medium 50% of the choline taken up was converted into ACh. The hemicholinium‐3 sensitive uptake of choline, the conversion of choline into ACh and the synthesis of total ACh, were stimulated about 7–8‐fold by potassium. It is concluded that in cortical slices from rat brain all choline used for the synthesis of ACh is supplied by the high‐affinity uptake system, of which the activity is geared to the rate of ACh synthesis.
ROLE OF MALATE TRANSPORT IN REGULATING METABOLISM IN MITOCHONDRIA ISOLATED FROM RABBIT BRAIN
Authors: Beck D. P. , Broyles J. L. , Von Korff R. W.
Published: October 2006

Abstract— The competition between citrate synthase and aspartate amino transferase for oxaloacetate raises the question of what process controls the availability of oxaloacetate. There are two possible limiting steps (in brain mitochondria pretreated with malonate and incubated in the presence of malate), malate translocation or malate dehydrogenase activity. Incubation of mitochondria with Triton X‐100 (100 ng/ml) resulted in an increased malate penetration as measured by a filtration method, but resulted in no stimulation of aspartate formation via aspartate aminotransferase. Inclusion of p‐trifluorometh‐oxycarbonyl‐cyanide phenylhydrazone (0.3 μm) along with Triton (100 ng/ml) in the incubation medium resulted in increased aspartate formation, presumably by raising the NAD+/NADH ratio and thereby increasing the activity of malate dehydrogenase. We conclude from the data described in this report that under the conditions employed here limitation in the availability of oxaloacetate determined by malate dehydrogenase activity controls mitochondrial metabolism and that the malate carrier is not rate limiting.
KINETICS OF BRAIN GLUTAMATE DECARBOXYLASE. INTERACTIONS WITH GLUTAMATE, PYRIDOXAL 5‐PHOSPHATE AND GLUTAMATE‐PYRIDOXAL 5‐PHOSPHATE SCHIFF BASE1
Authors: Bayón A. , Possani L. D. , Tapia M. , Tapia R.
Published: October 2006

Abstract— The kinetic behavior of glutamate decarboxylase from mouse brain was analyzed in a wide range of glutamate and pyridoxal 5′‐phosphate concentrations, approaching three limit conditions: (I) in the absence of glutamate‐pyridoxal phosphate Schiff base; (II) when all glutamate is trapped in the form of Schiff base; (III) when all pyridoxal phosphate is trapped in the form of Schiff base. The experimental results in limit condition (I) are consistent with the existence of two different enzyme activities, one dependent and the other independent of free pyridoxal phosphate. The results obtained in limit conditions (II) and (III) give further support to this postulation. These data show that the free pyridoxal phosphate‐dependent activity can be abolished when either all substrate or all cofactor are in the form of Schiff base. The free pyridoxal phosphate‐independent activity is also abolished when all substrate is trapped as Schiff base, but it is not affected by the conversion of free pyridoxal phosphate into the Schiff base. A kinetic and mechanistic model for brain glutamate decarboxylase activity, which accounts for these observations as well as for the results of previous dead end‐inhibition studies, is postulated. Computer simulations of this model, using the experimentally obtained kinetic constants, reproduced all the observed features of the enzyme behavior. The possible implications of the kinetic model for the regulation of the enzyme activity are discussed.
IMMUNOCHEMICAL STUDIES OF MAMMALIAN BRAIN ACETYLCHOLINESTERASE1,2
Authors: Greenberg A. J. , Parker K. K. , Trevor A. J.
Published: October 2006

Abstract— Specific antibodies were raised in rabbits to acetylcholinesterase (AChE) from bovine caudate nucleus and the‘native’(14S + 18S) and globular (11S) forms of AChE from eel electric tissue. All AChE preparations were purified by affinity chromatography to a specific activity of 100–400 mmol acetylthiocholine hydrolyzed/mg protein/h. Antigenic specificities of the different enzyme forms were studied by immunodiffusion, Immunoelectrophoresis and micro‐complement fixation. Minor differences in antigenic determinants were observed between the different molecular forms of electric tissue AChE. In crossover experiments using both eel AChE and bovine caudate AChE antisera there was complete absence of cross reactivity between the mammalian brain AChE and the different molecular forms of the electric tissue enzyme. Brain AChE activity was inhibited up to 50% in the presence of its antiserum.