bluuebunny/crossref_metadata_2025 huggingface dataset:
PHOSPHORYLATION OF NUCLEAR PROTEINS FROM RABBIT CEREBRUM, CEREBELLUM AND LIVER IN VITRO
Authors: Yanagihara T. , Oh'hara I. , Arvidson C. , Gintz J.
Published: December 2006

Abstract— Phosphorylation of nuclear protein was investigated with isolated nuclei from rabbit cerebral cortex, cerebellum and liver by using [γ‐32P]ATP. The results were compared with the previously reported findings on phosphorylation with tissue slices and [32P]phosphate. Cerebral cortex showed a very high level of phosphorylation, while liver showed the lowest, the difference being several fold in magnitude. With each tissue source, the extent of phosphorylation was maximum at incubation period for 2–3 min with steady decline afterwards. When nuclear proteins were further fractionated into 0.14m‐NaCl‐soluble, 0.25 n‐HCl‐soluble (mainly histone) and acidic phenol‐soluble proteins, NaCl‐soluble protein showed the highest phosphorylation while HCl‐soluble the lowest. The ratio among these tissue sources studied and the ratio among various protein fractions in each tissue source were strikingly similar to what had been shown with tissue slices. Further separation of acidic phenol‐soluble protein with polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate revealed retention of the characteristic difference of the pattern of phosphorylation between liver and the CNS tissue as having been observed with tissue slices, although phosphorylation of proteins with molecular weights of less than 40,000 was much reduced with the isolated nuclei. Although other methods with extracted protein kinase or chromatic protein fractions might be more desirable under ordinary situations, the system for nuclear protein phosphorylation with isolated nuclei and [γ‐32P]ATP may be useful under certain experimental conditions provided the incubation condition is carefully selected.
PROTEINS OF AXONAL TRANSPORT: GEL FILTRATION IN SODIUM DODECYL SULPHATE OF COMPONENTS WITH DIFFERENT SOLUBILITY CHARACTERISTICS
Authors: Karlsson J.‐O.
Published: December 2006

Abstract— Rapid axonal transport of proteins in retinal ganglion cells of the rabbit was studied following intraocular injection of labelled amino acids. It was found that rapidly transported material could be completely solubilized by sequential extraction with (a) isotonic buffer, (b) hypotonic buffer, (c) non‐ionic detergents (major part), (d) deoxycholate and (e) sodium dodecyl sulphate. All these fractions contained a major labelled macromolecular peak with a mol.wt. of 70,000–95,000 as determined by gel filtration in SDS. The molecular weight of this labelled peak seemed to be inconsistent with previous estimations based upon electrophoresis in sodium dodecyl sulphate.
SOLUBLE AND MEMBRANE BOUND CARBONIC ANHYDRASES FROM RAT CNS: REGIONAL DEVELOPMENT
Authors: Sapirstein Victor S. , Lees Marjorie B. , Trachtenberg Michael C.
Published: December 2006

Abstract— The distribution of the soluble, membrane bound and myelin carbonic anhydrase in different regions of the rat CNS was examined as a function of age. A neuraxial progression from spinal cord to upper brain stem was observed for all three enzyme fractions in the 90 day old rat: upper brain stem > lower brain stem and cerebellum > spinal cord. The membrane bound fraction accounted for close to 60% of the total carbonic anhydrase in all regions except the cerebellum where it accounted for only 40%. The developmental pattern of the total membrane bound and soluble fractions were virtually parallel in all regions studied suggesting that they are derived from a common enzyme pool. The myelin enzyme accounts for a small but significant part of the membrane bound fraction and is present at adult levels by 16 days of age indicating it is an early and specific myelin component.
GALACTOCEREBROSIDE SULFOTRANSFERASE: FURTHER CHARACTERIZATION OF THE ENZYME FROM RAT BRAIN
Authors: Tennekoon G. , McKhann G. M.
Published: December 2006

Abstract— Myelin has an unusual lipid composition, being particularly rich in sulfatide. This lipid is synthesized by the transfer of sulfate from phosphoadenosine phosphosulfate to galactocerebroside, catalyzed by galactocerebroside sulfotransferase. This paper describes a sensitive assay for the sulfotransferase (capable of measuring activity in as little as 10 μg of extracted rat brain protein) so that this enzyme can be readily investigated in isolated cells, or the small amounts of tissue available in developing animals. Both manganase (20 mm) and thiol reagents were required for optimal activity. This assay was used to monitor the purification of the sulfotransferase from rat brain. Extraction of the enzyme from crude homogenates required the nonionic detergent, Triton X‐100, at pH 7–7.5. Removal of Triton X‐100 from the extracted enzyme resulted in a soluble but less active enzyme, the activity of which could then be restored with detergents. Stability of the detergent‐extracted enzyme was investigated, and even at —40°C there was a 20% loss of activity over 10 days. By standard procedures 500‐fold purification of the enzyme has been achieved.
IN VIVO INHIBITION OF RAT BRAIN PROTEIN SYNTHESIS BY d‐AMPHETAMINE
Authors: Roel L. E. , Moskowitz M. A. , Rubin D. , Markovitz D. , Lytle L. D. , Munro H. N. , Wurtman R. J.
Published: December 2006

Abstract— Between 1 and 4 h after rats received a single injection of d‐amphetamine (15 mg/kg)(when brain polysomes are known to be disaggregated), the in vivo incorporation of [14C]lysine into trichloroacetic acid‐precipitable brain protein was reduced by 28–48%. Incorporation of the 14C label into the protein present in a 100,000 g supernatant extract of whole brain was similarly reduced (by 44%). Amphetamine administration suppressed protein synthesis in rat cerebral cortex, cerebellum, hypothalamus, striatum, and brainstem to an equivalent extent. The drug did not significantly affect lysine pool sizes measured in these brain regions; thus the reduced incorporation of labeled lysine was not the result of an isotope dilution effect. We therefore conclude that the brain polysome disaggregation resulting from amphetamine administration is associated with decreased in vivo synthesis of some brain proteins.
Phosphorylation of Endogenous Proteins by Adenosine 3′:5′‐Monophosphate‐Dependent Protein Kinase in Mouse Neuroblastoma Cells
Authors: Prashad Nagindra , Evetts Curtis , Wischmeyer Betsy
Published: December 2006

Abstract: Increased intracellular adenosine 3′:5′‐monophosphate (cAMP) levels and activation of cAMP‐dependent protein kinases (ATP:protein phosphotransferase, EC 2.7.1.37) in vivo were correlated in mouse neuroblastoma cells grown in the presence of 1 mM‐6N.O2‐dibutyryl 3′:5′‐monophosphate (Bt2cAMP). The time course for activation showed that cAMP‐dependent protein kinases were activated by 30 min. A heat‐stable inhibitor protein inhibited a majority of activated cAMP‐dependent protein kinase. Activation of cAMP—dependent protein kinase caused additional phosphorylation of proteins when compared with untreated control cells, as demonstrated by endogenous phosphorylation of proteins in vitro using [γ‐32P]ATP and analysis by two—dimensional polyacrylamide gel electrophoresis. The phosphorylation data show selective phosphorylation of specific proteins by cAMP‐independent and cAMP‐dependent protein kinase. Among the proteins in the postmitochondrial supernatant fraction phosphorylated by cAMP‐dependent protein kinases, two proteins with a molecular weight of 43,000 were heavily phosphorylated. It is suggested that phosphorylation of cellular proteins by cAMP‐dependent protein kinases might be involved in the cAMP‐modulated biochemical changes in neuroblastoma cells.
The Synthesis of Neuroactive Amino Acids from Radioactive Glucose and Glutamine in the Rat Retina: Effects of Light Stimulation
Authors: Voaden Mary J. , Morjaria Bharti
Published: December 2006

Abstract: The effect of light stimulation in vitro on the labelling of neuroactive amino acids derived from [14C]glucose or [14C]glutamine in the rat retina has been studied. [14C]Glutamine, at 600 μM, provided about 50% of the tissue pools of glutamate, aspartate, and GABA; and the labelling of these decreased on light stimulation, both in the photoreceptor cells (glu and asp) and in the inner retina (glu, asp, and GABA). In contrast, there were no significant changes in the entry of label derived from [14C]glucose, although similar trends were apparent in the data obtained for the photoreceptor cell layer. The pools may, therefore, be separate. Other results support the contention that glucose is the principal energy source for the retina, its entry into non‐amino acid derivatives being decreased on light stimulation.
Substrate Selectivity of Type A and Type B Monoamine Oxidase in Rat Brain
Authors: Kinemuchi Hiroyasu , Wakui Yoshiko , Kamijo Kazuya
Published: December 2006

Abstract: Use of the irreversible inhibitors clorgyline and deprenyl showed that rat brain mitochondria contain type A and type B monoamine oxidase (MAO). Tyramine is a substrate for both types of MAO, whereas serotonin is a preferential substrate for type A MAO. In contrast to MAO in other tissues, type A MAO in brain tissue oxidizes β‐phenylethylamine (PEA) at high concentrations (0.5 and 1.0 mM). The proportions of type A and type B MAO activities in the mitochondria estimated from the double‐sigmoidal inhibition curves of tyramine oxidation were about 70:30 irrespective of the concentration of tyramine. With PEA as substrate, the ratios of type A to type B activities were found to increase from low values at low concentrations to about 1 at 0.5‐1.0 mM‐PEA, and even higher at further increased concentrations of PEA. At very low (0.01 mM) and high (10.0 mM) concentrations of PEA, single‐sigmoidal curves were obtained; with the high PEA concentration the activity was highly sensitive to clorgyline, whereas with the low concentration it was highly sensitive to deprenyl. In deprenyl‐pretreated mitochondrial preparations, all the remaining activity towards 0.5‐1.0 mM‐PEA was shown to be highly sensitive to clorgyline, demonstrating that this activity was indeed due to oxidation by type A MAO. The opposite result was obtained with deprenyl as inhibitor of clorgyline‐pretreated preparations, demonstrating that PEA at this concentration was also oxidized by type B MAO in rat brain mitochondria. The K3 values of type A and type B MAO for PEA were significantly different. On Lineweaver‐Burk analysis, plots with PEA as substrate for type A MAO in a deprenyl‐treated preparation were linear over a wide concentration range, whereas those for type B MAO in a clorgyline‐treated preparation were not linear, but showed substrate inhibition at higher concentrations of the substrate. It is concluded from the present findings that the effect of the substrate concentration must be considered in studies on the characteristics of multiple forms of MAO in various organs and species.
The γ‐Aminobutyrate Content of Nerve Endings (Synaptosomes) in Mice After the Intramuscular Injection of γ‐Aminobutyrate‐Elevating Agents: A Possible Role in Anticonvulsant Activity
Authors: Wood J. D. , Russell M. P. , Kurylo E.
Published: December 2006

Abstract: The intramuscular administration of L‐cycloserine, gabaculine, and aminooxyacetic acid caused significant, time‐dependent increases in the γ‐aminobutyric acid (GABA) content of both whole brain and synaptosomalenriched preparations obtained from the tissue, a linear relationship being observed between the two parameters. In contrast, the administration of hydrazine resulted in a large increase in whole brain GABA level, with little change in the synaptosomal GABA content. The key factor in these different responses appeared to be the degree of inhibition of glutamic acid decarboxylase by the drugs. Pretreatment of mice with the GABA‐elevating agents resulted in a delay in the onset of seizures, which was related directly to the increase in synaptosomal GABA content. Although the seizures were delayed, they occurred while the GABA content of nerve endings (synaptosomes) was above that in preparations from untreated animals. The decrease in GABA content at the onset of seizures, expressed as a percentage of the level at the time of injection of the convulsant agent, was, however, reasonably constant. A hypothesis to explain these results is proposed.
Effect of Stimulation on the Incorporation of 14C from Glial and Neuronal Specific Substrates into Brain Proteins In Vivo and In Vitro
Authors: Abdul‐Ghani A.‐S. , Boyar M. M. , Coutinho‐Netto J. , Bradford H. F.
Published: December 2006

Abstract: The incorporation of amino acids into brain proteins following brachial plexus stimulation (BPS) was studied in anaesthetised Sprague‐Dawley rats following injection of radioactive precursors of both neuronal and glial compartments. Following intraperitoneal injection of [14C]glucose, which is the major neuronal pool precursor, BPS resulted in a significant increase of 379% (P± 0.001) in the incorporation of carbon from [14C]glucose into TCA‐insoluble proteins in the contralateral sensorimotor cortex as compared with the ipsilateral area of the same animal. This increase was abolished totally when tetrodotoxin (10 μg ml‐1) was applied topically to the surface of the stimulated area. Following intraperitoneal injection of [14C]acetate, which is considered to be mainly a glial cell precursor, the same afferent electrical stimuli caused a significant decrease of 21% in the incorporation of amino acids into proteins in the stimulated versus unstimulated sensorimotor cortex. With [4‐3H]phenyl‐alanine or [l‐14C]leucine as precursors a significant decrease (12%) or no change was recorded, respectively. A similar decrease in protein synthesis in the stimulated sensorimotor cortex was achieved using different routes of injection. No significant changes were observed in the ratio of the specific radioactivities of the total amino acids of the two hemispheres using either precursor. In vitro, synaptosomes showed a large increase in incorporation into proteins after treatment with electrical pulses, both with [14C]glucose and with [U‐14C]acetate as precursors.
Alterations in β1‐ and β2‐Adrenergic Receptor Density in the Cerebellum of Aging Rats
Authors: Pittman R. N. , Minneman K. P. , Molinoff P. B.
Published: December 2006

Abstract: The densities of β1, and β2‐adrenergic receptors were determined in homogenates of cerebral cortex and cerebellum of rats between 3 and 14 mo of age. No change in either receptor population occurred in the cortex during this period. In the cerebellum, a 20–25% decrease in the density of β2, receptors and a 3509% increase in the density of β1, receptors occurred. The increase in β1 receptors in the cerebellum may be the result of a decrease in the function of the noradrenergic projections from the locus coeruleus which synapse on cerebellar Purkinje cells.
Stability Properties of Activated Tryptophan Hydroxylase from Rat Midbrain
Authors: Vitto Anthony , Mandell Arnold J.
Published: December 2006

Abstract: Time courses of the activation‐inactivation sequence in rat midbrain tryptophan hydroxylase after preincubation with calcium, ATP + MgCl2, or sulfhydryl reagents and after freezing and thawing suggest that the activated enzyme is more vulnerable to loss of activity. The sequence induced by calcium was prevented by the protease inhibitor leupeptin, and an accelerated decline in activity after activation by ATP + MgCl2 was reduced greatly by increasing levels of tetrahydrobiopterin (BH4) cofactor. The effects of calcium and ATP + MgCl2 were additive, which suggests independent mechanisms. The findings suggest that time courses of enzyme activation and inactivation processes may offer a useful way to study the influence of a range of effectors on tryptophan hydroxylase function.
Long‐Term Treatment of Rats with Morphine Reduces the Activity of Messenger Ribonucleic Acid Coding for the β‐Endorphin/ACTH Precursor in the Intermediate Pituitary
Authors: Höllt Volker , Haarmann Ingeborg , Herz Albert
Published: December 2006

Abstract: Chronic administration of morphine to rats for a period of 4 weeks resulted in a 50‐60% decrease in the tissue concentrations of β‐endorphin and in the in vitro release from the neurointermediate pituitary. Incorporation of [3H]phenylalanine into isolated intermediate/posterior pituitaries in vitro revealed a reduction in the amount of label incorporated into the β‐endorphin/ ACTH precursor to a similar extent (about 45%), but essentially no effect on the conversion of the precursor into β‐lipotropin and β‐endorphin. Extraction of mRNA from intermediate/posterior pituitaries followed by cell‐free translation in a reticulocyte system showed no significant decrease in the total level of translatable mRNA. In contrast, the content of translatable mRNA coding for the β‐endorphin/ACTH precursor was significantly reduced by 50‐60%. Thus, long‐term treatment with morphine appears to depress β‐endorphin formation in the rat intermediate pituitary at the pretranslational level by markedly decreasing the activity of mRNA coding for the β‐endorphin/ACTH precursor without any alteration in the processing of this precursor.
Characterization of Chick Gizzard Extract That Promotes Neurite Outgrowth in Cultured Ciliary Neurons
Authors: Miki Naomasa , Hayashi Yokichi , Higashida Haruhiro
Published: December 2006

Abstract: Chicken gizzard extract contains a macromolecule(s) that promotes the neurite outgrowth of dissociated neurons from the ciliary ganglia (CG) of chick embryos. The factor in gizzard extract was partially purified and estimated to be about 12S (M.W. 200,000‐300,000) on sucrose density gradient centrifugation. The neurite outgrowth of CG neurons by the factor strictly depends on the embryonal age. The maximal neurite outgrowth was observed when CG neurons were dissociated from the embryos younger than 10 days. After that time the response of CG neurons to the factor rapidly declined and was almost lost at day 14. The amount of factor in the gizzard began to increase rapidly from 12‐day‐old embryo and reached the maximal level at day 16, and thereafter a fairly steady level was maintained. When CG neurons were co‐cultured with rat myotubes, the ratio of muscle cells with synaptic responses (miniature end‐plate potentials) was significantly higher in the presence of gizzard factor than its absence. The results suggest that this factor acts as an external signal on CG neurons to form synaptic connections in vivo.
Increased Noradrenergic Metabolism in the Cerebellum of the Mouse Mutant Dystonia Musculorum
Authors: Riker Donald Kay , Messer Anne , Roth Robert H.
Published: December 2006

Abstract: The neurological mouse mutant dystonia musculorum exhibits bizarre appendicular and truncal dystonia without known cerebellar histopathology. We evaluated striatal dopamine and cerebellar norepinephrine metabolism in this mutant and compared the results with those obtained in wild‐type BALB/c and B6C3 controls. Tyrosine hydroxylase activity and dopamine metabolite levels (homovanillic acid and 3,4‐dihydroxyphenylacetic acid) in the striatum of the mutant were similar to controls. Tyrosine hydroxylase activity and the steady‐state level of 3‐methoxy‐4‐hydroxyphenethyleneglycol, a metabolite of norepinephrine, in the cerebellum were 38% and 42‐66%, respectively, greater in the mutant. However, the level of norepinephrine was similar (∼350 ng/g). Further, a Purkinje cell‐specific marker, cGMP‐dependent protein kinase, was unchanged in the mutant and no Purkinje cell pathology was observed with light microscopy. The lack of Purkinje cell derangement and similar levels of cerebellar norepinephrine and cGMP‐dependent protein kinase activity suggest that increased norepinephrine metabolism in the cerebellum of this mutant is not a morphological response to gross target cell loss during morphogenesis. The observed changes may be a reaction to abnormal impulse traffic or altered input/output pathways to the mutant cerebellum during its development.
Role of Elongation Factor 1 in the Translational Control of Rodent Brain Protein Synthesis
Authors: Vargas Rocío , Castañeda Mario
Published: December 2006

Abstract: The translational control of protein synthesis during early postnatal neural development and aging was examined in the mouse and the rat. The activity of brain elongation factor 1 (bEF‐1) was found to decrease exponentially with age and to decline parallel to the age‐dependent decrease in total protein synthesis in both rodents. This decrement in bEF‐1 activity fell within the range of reported age‐related decreases in protein synthesis in in vitro systems. The factor was present in multiple forms; the lighter species predominated in older animals, whereas the young light form apparently disappeared with increasing age, and was replaced by others arising from the heavy form. Elongation factor 1 derived from young brains functioned as a rate‐limiting component in polypeptide synthesis in previously saturated adult systems. The data suggest that bEF‐1 has an important modulatory effect on total brain protein synthesis.
The Biosynthesis of Polyamines in the Brain of Audiogenic Seizure‐Susceptible and ‐Resistant Deermice
Authors: Porta Raffaele , Doyle Richard L. , Tatter Stephen B. , Wilens Timothy E. , Schatz Robert A. , Sellinger Otto Z.
Published: December 2006

Abstract: The biosynthesis of polyamines was investigated in the brains of the audiogenic seizure‐susceptible (SS) mutant and the wild‐type, seizure‐resistant (SR) deermouse Peromyscus maniculatus bairdii. For this purpose a new, rapid, and economical high pressure liquid chromatography (HPLC) procedure for the quantitation of putrescine, spermidine, and spermine was developed. Benzoyl derivatives of the polyamines, prepared from a crude brain supernatant, were ether extracted and, following removal of the ether, were separated and quantitated by HPLC. The high sensitivity of the method allows quantitation of putrescine in 50 mg and of spermidine and spermine, in as little as 2‐2.5 mg, of brain tissue. No differences were found in endogenous levels of the 3 polyamines in brains of SS vs SR deermice. Using [14C]putrescine as a polyamine precursor, we found the specific radioactivity of spermidine to be lower in the SS than in the SR brains following a 1 h intraventricular (i.vt.) pulse. No such differences were noted if [3,4‐14C]methionine was used as the polyamine precursor. To test whether the flux of methionine through the transmethylation pathway was also different in SS and SR deermouse brain, we administered [1‐14C]methionine (i.vt.) (1 h pulse). Even though the brains of SS animals contained higher methionine and lower S‐adenosyl‐l‐methionine (AdoMet) levels than the SR brains, the specific radioactivities of methionine and AdoMet were, respectively, lower and higher in SS compared to SR brains. The latter results are in agreement with our previous findings of an accelerated utilization of AdoMet in brains of Swiss‐Webster mice following administration of the chemical convulsant l‐methionine‐d,l‐sulfoximine (MSO). Taken together, the data suggest that the SS condition, whether genetically determined (as in the SS deermouse) or chemically elicited (as after MSO), correlates positively with higher than normal rates of conversion of methionine to brain AdoMet and leads to an enhanced rate of utilization of AdoMet via the transmethylation pathway.
Estimation of the p and m Isomers of Hydroxyphenylacetic Acid in Mouse Brain by a Gas Chromatographic Procedure: Their Regional Distribution and the Effects of Some Drugs
Authors: McQuade P. S. , Juorio A. V. , Boulton A. A.
Published: December 2006

Abstract: The mouse brain contains 12.5 and 4.1 ng/g of p‐ and m‐hydroxyphenylacetic acids, respectively. The hydroxyphenylacetic acids were isolated by chromatography on DEAE‐Sephadex A‐25 and quantitated as their pentafluoropropionyl and hexafluoropropanol esters by use of a gas chromatograph equipped with an electron‐capture detector. The highest concentrations of p‐ or m‐hydroxyphenylacetic acids were observed in the caudate nuclei (27.9 and 8.7 ng/g, respectively) and olfactory tubercles (20.2 and 5.3 ng/g, respectively). The identities of the p‐ and m‐hydroxyphenylacetic acids were further confirmed as a consequence of the reductions observed following monoamine oxidase inhibition or the increases observed in the appropriate acid following the parenteral administration of p‐ or m‐tyramine.
Membrane Populations of Bovine Choroid Plexus: Separation by Density Gradient Centrifugation in Modified Colloidal Silica
Authors: Mamelok Richard D. , Macrae Donald R. , Benet Leslie Z. , Prusiner Stanley B.
Published: December 2006

Abstract: The choroid plexus is intimately involved in the production and regulation of the cerebrospinal fluid. Populations of surface membranes from this epithelial tissue were separated by density gradient centrifugation by use of modified colloidal silica (Percoll). A fraction of heavy microsomes (P3) containing plasma membranes was prepared by differential centrifugation. Membranes in fraction P3 were mixed with a given concentration of Percoll and density gradients generated during centrifugation. When fraction P3 was mixed with 20% (v/v) Percoll and centrifuged at 20,000 r.p.m. for 1 h in a 50.2 Ti fixed‐angle rotor, membranes containing alkaline phosphatase (AP) were found at a density of 1.037 g/cm3 while those containing NaK ATPase were found at 1.047 g/cm3. With more shallow density gradients using 12% and 14% Percoll, a broad shoulder of AP activity became manifest at densities greater than 1.060 g/cm3 suggesting multiple populations of membranes containing AP. Membranes containing AP could also be separated from membranes containing γ‐glutamyl transpeptidase (γ‐GTP); this separation was most pronounced in 12% Percoll. The activity of γ‐GTP could not be separated from activity of NaK ATPase. Total protein was distributed broadly throughout the gradients. Studies have been undertaken to compare the behavior of choroidal membranes in Percoll gradients with that of renal membranes because the biochemical anatomy of the kidney has been extensively studied. In contrast to choroidal membranes, renal membranes with NaK ATPase activity were found to have densities lower than those membranes with AP. Thus, the distribution of membrane‐bound enzymes from kidney in a Percoll gradient was exactly the opposite of that observed for these same enzymes from choroid plexus. In addition, unlike the γ‐GTP activity of choroid plexus, γ‐GTP from kidney could be separated from the activities of both alkaline phosphatase and NaK ATPase. These marked differences in membrane populations between choroid plexus and kidney as defined by Percoll density gradient centrifugation analyses are presumably reflective of differences in the functions of the two epithelial tissues.
Characteristics of Dihydroxyphenylalanine/5‐Hydroxytryptophan Decarboxylase Activity in Brain and Liver of Cat
Authors: Bouchard Sylvie , Bousquet Christiane , Roberge Andrée G.
Published: December 2006

Abstract: Dihydroxyphenylalanine/5‐hydroxytryptophan (DOPA/5‐HTP) decarboxylase activity varied widely in different parts of the CNS, being highest in the neostriatum and lowest in the frontal cortex. The addition of 2.5 μm‐pyridoxal 5′‐phosphate (PLP), the coenzyme, increased enzyme activity in brainstem and liver, while higher concentrations led to a decrease in activity. In brainstem, the addition of 1000 μm PLP shows activity similar to that obtained without exogenous PLP. The effects of different monoamine oxidase (MAO) inhibitors on decarboxylase activity were demonstrated. Iproniazid phosphate and harmaline significantly decreased the decarboxylation in liver and brainstem, while pargyline inhibited only liver decarboxylation. Some decarboxylase inhibitors such as RO4–4602 and α‐methyl DOPA, as well as piribedil, a dopaminergic receptors agonist, were added in vitro to measure their action on decarboxylase with or without exogenous PLP or with double concentrations of substrate (5‐HTP). Piribedil (5000 μm) affected the enzymic reaction and triggered a higher inhibition in liver. Inhibition in brainstem needed less RO4–4602 (50 μm) than in liver (300 μm). Addition of PLP did not reverse this inhibition, while doubling the concentration of 5‐HTP nullified the inhibitory effect in liver only. Inhibition induced by α‐methyl DOPA (5 μm) was easily reversed by doubling the concentration of substrate. However, the presence of exogenous PLP restored the enzymic activity in liver only. We conclude from this work thus that the enzyme can decarboxylate its substrate without exogenous PLP, that MAO inhibitors might inhibit decarboxylase activity, and that decarboxylase inhibitors react differently when brain and liver are used as enzymic source. PLP seems to act as a protective agent on the active site of the enzyme in the brainstem and preferentially with the substrate in the liver.
Enzyme Immunoassay of the Myelin Basic Protein
Authors: Shinomiya Y. , Kato N. , Imazawa M. , Miyamoto K.
Published: December 2006

Abstract: An enzyme immunoassay using a double‐antibody solid‐phase technique for myelin basic protein (MBP) has been developed. Antisera were prepared by immunizing rabbits with the purified MBP from chick brain. The conjugation of MBP with horseradish peroxidase was performed by the periodate oxidation method in triethanolamine‐acetate buffer (pH 8.5). The sample, antiserum, and conjugate were incubated at 4°C for 16 h, after which the insoluble second antibody was added and the reaction mixture was incubated at 4°C for 3 h. The peroxidase activity of the insoluble conjugate was assayed fluorometrically with hydrogen peroxide and 3‐(p‐hydroxyphenyl)propionic acid as substrates. The method had an analytical range from 50 pg to 1 ng (from 2.3 × 10−15 to 4.5 × 10−14 mol). The within‐assay coefficient of variation (CV) was between 4 and 11% and the between‐assay CV for 200 and 400 pg of MBP was 5.5 and 7.1%, respectively. A weak cross‐reactivity was observed between chick MBP and bovine MBP, while no reactivity was shown with calf thymus histone. The MBP content of the brain during development increased markedly from the 3rd embryonic week to the 3rd post‐hatch week (from 0.01 to 2.4 mg/g of fresh tissue), and the adult level was 3.2 mg/g of fresh tissue.
Glycosylceramide Synthesis in the Developing Spinal Cord and Kidney of the Twitcher Mouse, an Enzymatically Authentic Model of Human Krabbe Disease
Authors: Kodama Soichi , Igisu Hideki , Siegel Donald A. , Suzuki Kunihiko
Published: December 2006

Abstract: UDP‐galactose:ceramide galactosyltransferase activity was assayed in the spinal cord and kidney of the recently discovered neurological mutant, the twitcher mouse, which is an enzymatically authentic model of human globoid cell leukodystrophy (Krabbe disease). The activity in the spinal cord was essentially normal during the early myelination period up to 15 days. There was a slight reduction at 20 days. At 25 and 33 days, the galactosyltransferase activity was drastically reduced compared to controls. In contrast, the galactosyltransferase activity in the kidney of twitcher mice remained normal throughout the developmental stages examined. Activity of the control enzyme UDP‐glucose:ceramide glucosyltransferase was always normal in both the spinal cord and kidney. Thus, reduction of galactosylceramide synthesis occurs in the CNS secondarily to the pathological alteration of the oligodendroglia. No such reduction occurs in the kidney, at least for the last step of galactosylceramide synthesis. Reduced synthesis as the result of metabolic regulation in the presence of the catabolic block is therefore unlikely to be the cause of the lack of abnormal accumulation of galactosylceramide in the kidney of patients with globoid cell leukodystrophy.
A Radioimmunoassay for the Myelin‐Associated Glycoprotein
Authors: Johnson David , Quarles Richard H. , Brady Roscoe O.
Published: December 2006

Abstract: The myelin‐associated glycoprotein (MAG) was purified from rat brain and radioiodinated with Bolton‐Hunter reagent for use in a double‐antibody radioimmunoassay. The conditions of the assay were adjusted to measure between 2 and 30 ng of MAG. The antigenic sites of MAG in tissue samples were exposed by solubilization in 1% (wt/vol) sodium dodecyl sulfate (SDS), and the final assay was done in a mixture of 0.25% SDS and 0.25% Triton X‐100. The presence of the Triton X‐100 overcame the inhibitory effect of SDS alone on the immune reactions. Application of the assay to whole homogenates of developing rat brain revealed the expected increase of MAG with the progression of myelination. Adult brain homogenate and purified myelin contained 2.7 and 7.4 μg of MAG/mg protein, respectively. Sciatic nerve contained a lower level of MAG, and cross‐reacting material was not detected in nonneural tissues. This assay makes possible for the first time the analysis of MAG in whole tissue without prior myelin isolation or glycoprotein separation.
Effects of Na+, Ca2+, and Acetylcholine on Phosphoinositide‐ and ATP‐Phosphate Turnover in 32P‐Labeled Rabbit Iris Smooth Muscle
Authors: Akhtar Rashid A. , Abdel‐Latif Ata A.
Published: December 2006

Abstract: Parallel studies were carried out in the rabbit iris on (a) the effects of Na+ and/or Ca2+ on the acetylcholine‐stimulated 32P labeling of phosphatidic acid (PA) and phosphatidylinositol (PI) and the breakdown of polyphosphoinositides (poly PI), and (b) the effects of these cations on the specific radioactivity of [γ‐32P]ATP. Incorporation of 32Pi into ATP and phosphoinositides is time‐dependent, and it is remarkably dependent upon Na+ concentration in the incubation medium. The Na+ effect is reversible. Calcium ion, in the absence of Na+, had no effect on the specific radioactivity of ATP in 32P‐labeled iris muscle; however, it moderately stimulated the 32P labeling of PA and PI and the breakdown of poly PI. In contrast, the addition of Na+, in the presence or absence of Ca32, significantly reduced the specific radioactivity of ATP and 32P labeling of phospholipids in the 32P‐labeled iris muscle. Acetylcholine had no measurable effect on the specific radioactivity of ATP. Furthermore, the neurotransmitter stimulated the 32P labeling of PA and PI and the breakdown of poly PI in the 32P‐labeled muscle only in the presence of both Na+ and Ca+. These data provide additional support for the concept that in the rabbit iris receptor‐activated Ca2+ fluxes mediate or precede the effects of α‐adrenergic and cholinergic muscarinic agents on phosphoinositide breakdown into 1,2‐diacylglycerol and inositol phosphates and that restoration of the polar head groups to the 1,2‐diacylglycerol (i.e., the recovery stage) is probably associated with Na+ outflux, via the Na+‐pump mechanism.
Phosphoproteins of the Adrenal Chromaffin Granule Membrane
Authors: Burgoyne Robert D. , Geisow Michael J.
Published: December 2006

Abstract: A fraction of chromaffin granule membranes contained a number of substrates for endogenous protein kinase activity as well as endogenous phosphatase activity. The major 32P‐labelled polypeptide of molecular weight 43,000 appeared to be the α‐subunit of pyruvate dehydrogenase of residual mitochondria. Several polypeptides showed cyclic AMP stimulation of phosphorylation of which the major polypeptide of molecular weight 59,000 shows half‐maximal phosphorylation with 0.49 μM cyclic AMP. The phosphorylation of several other polypeptides is inhibited at high cyclic AMP concentrations. From studies with immunoprecipitation and two‐dimensional electrophoresis it was found that α‐ and β‐tubulin and actin were absent from the granule membranes. However 32P labelling of a proportion of the copies of dopamine‐β‐hydroxylase was demonstrated. The majority of the substrates for endogenous protein kinase activity are probably on the cytoplasmic side of the granule membrane.
The Independency of Choline Transport and Acetylcholine Synthesis
Authors: Marchbanks R. M. , Kessler P. D.
Published: December 2006

Abstract: The coupling of choline transport to acetylcholine synthesis has been investigated by measurement of the isotopic dilution of a pulse of [3H]choline during its incorporation into the recently synthesised acetylcholine of cerebral cortex synaptosomes. Recently synthesised acetylcholine was identified as that containing 14C‐labelled precursors introduced by a preincubation before the pulse. When [14C]glucose was used to label acetyl‐CoA coupling ratios (calculated as the inverse of the dilution of extracellular [3H]choline during its incorporation into [3H]acetylcholine) of about 0.05–0.2 were found at a choline concentration of 1 μM, rising to 0.5 at choline concentrations of 10–50 μM. Experiments using [14C]choline as a precursor gave similar results, and it was shown that the isotopic dilution did not occur extrasynaptosomally and was not affected by low glucose concentrations. Coupling ratios were always less than unity and rose as the choline concentration increased. It is concluded that choline transported into the nerve terminal has no privileged access to choline acetyltransferase. The results can be explained by a rate‐controlling transport of choline into the terminal followed by its rapid acetylation rather than any linkage or coupling of the two processes.
Developmental Changes in Glycolipid Synthesizing Enzymes in the Brain of a Myelin‐Deficient Mutant Wistar Rat
Authors: Hof Liselotte , Csiza Charles K.
Published: December 2006

Abstract: Changes in the activities of UDP‐galactose:ceramide galactosyl‐transferase (CGaIT, EC 2.4.1.45), UDP glucose:ceramide glucosyltransferase (CGIcT, EC 2.4.1.80) and 3′‐phosphoadenosine‐5′‐phosphosulfate (PAPS): galactosylceramide 3′‐sulfotransferase (EC 2.8.2.11) over the myelinating period between 12 and 25 days were studied in the brains of control and myelin‐deficient rats. Although the activity of galactosyltransferase with ceramides containing hydroxy fatty acids quadrupled in normal male litter‐mates between 14 and 20 days, hardly any increase was observed in the mutant and the activity was less than 10% of control above 20 days of age. With normal fatty acid containing ceramides as acceptors, the activity decreased from 83% of the control at 12 days to ∼ 30% after 20 days. Sulfotransferase activity also did not show the normal increase during the 3rd week of life and declined from 60% to 22%. Glucosyltransferase and lysosomal hydrolases in brain and ceramide galactosyltransferase in sciatic nerves appeared to be normal. These results suggest close similarities to the jimpy mutant mouse in which myelin deficiency is also inherited as an x‐linked recessive trait.
Ethylenediamine and GABA Potentiation of [3H]Diazepam Binding to Benzodiazepine Receptors in Rat Cerebral Cortex
Authors: Morgan P. F. , Stone T. W.
Published: December 2006

Abstract: Specific binding of [3H]diazepam at a free concentration of 2 nM was found to be maximally potentiated by 117% in Tris‐HCl buffer and 160% in Tris‐citrate buffer by ethylenediamine (EDA), but only at relatively high concentrations of EDA (ED50= 5 × 10−5M), although this potentiation was susceptible to a low dose (6 μM) of bicuculline. Dose‐response curves show that EDA differs from GABA with respect to both potency and efficacy. In additivity experiments no evidence was found that EDA could act as a partial agonist at GABA receptors, and it was concluded that EDA and GABA apparently do not potentiate [3H]diazepam binding by acting on the same receptor. Scatchard analysis lends support to this hypothesis, indicating that the potentiation of [3H]diazepam binding by 3.16 × 10‐3M EDA is due to an increase in receptor number (from 930 to 1170 fmol/mg protein) and not receptor affinity (remaining constant about 20 nM). Subsequent studies showed the potentiation to be reversible. It is concluded that EDA can act on the GABA‐benzodiazepine receptor ionophore complex but that this is probably not a direct action on the GABA receptor. It is suggested that EDA can be used to differentiate GABA receptors linked to benzodiazepine receptors from those not so linked.
Differential Effects of Long‐Term Electroconvulsive Shock on Brain Levels of Enkephalin and Humoral‐Endorphin
Authors: Same Y. , Weissman B. A. , Urca G.
Published: December 2006

Abstract: Electroconvulsive shock (ECS) administrations repeated for 10 consecutive days cause an elevation in the opioid content of the rat brain. Two different endogenous opioids, enkephalin and humoral‐endorphin, undergo independent changes that differ in both their time course and intracerebral localization. These metabolic changes parallel long‐term behavioral modifications such as the development and dissipation of tolerance to the analgesic effect of ECS. The activation of two different, independent, endogenous opioid systems by ECS is in agreement with previous behavioral and pharmacological studies.
Respective Roles of Ammonia, Amino Acids, and Medium‐Sized Molecules in the Pathogenesis of Experimentally Induced Acute Hepatic Encephalopathy
Authors: Denis J. , Delorme M. L. , Boschat M. , Nordlinger B. , Opolon P.
Published: December 2006

Abstract: Ammonia, amino acids (AA), and middle molecules (MM) have been implicated in the pathogenesis of experimentally induced acute hepatic coma in the pig. Hemodialysis (HD) using either a low‐ (Cuprophan = CU) or a high‐permeability (polyacrylonitrile = AN 69°) membrane has demonstrated the role of MM. Selective hemodialysis (SHD) of AA or NH3 and MM was performed by adding either NH3 (group I) or AA (group II) to the dialysate during AN 69 HD; for MM, SHD only was performed by adding NH3 and AA to the dialysate (group III). In group I the brain levels of tyrosine were similar to those in undialyzed animals with decreased striatal dopamine and decreased norepinephrine in the midbrain only. Brain tryptophan was higher than normal, but brain levels of 5‐hydroxytryptamine and 5‐hydroxyindoleacetic acid (5‐HT, 5‐HIAA) were within normal limits. In group II, despite an efficient NH3 clearance, brain NH3 levels were as high as in group I and did not correlate with plasma levels. Brain tyrosine (despite tyrosine overload of the dialysate) was lower than in group I; striatal dopamine decreased (but to a lesser extent than in group I), and norepinephrine was normal. Brain tryptophan was higher than normal, with an increase in brain 5‐HT and 5‐HIAA. In group III, results were similar to group I, except for a limited increase of 5‐HT in the pons. Brain octopamine levels increased only in undialyzed and CU‐HD animals, demonstrating a specific relation with MM. These experiments demonstrate the interrelationship between NH3 and neutral AA with regard to passage through the blood‐brain barrier and to intracerebral metabolism.
Rapid Axonal Transport of Glycerophospholipids in Regenerating Hypoglossal Nerve of the Rabbit
Authors: Alberghina M. , Viola M. , Giuffrida A. M.
Published: December 2006

Abstract: The intraaxonal transport of phospholipids in regenerating hypo‐glossal nerve of the rabbit was investigated by administration of labeled lipid precursors into the medulla oblongata. At various time intervals after crushing the left hypoglossal nerve at the level of the digastric muscle, a mixture of 60 MCi of [2‐3H]glycerol and 15 /μCi of [1‐14C]palmitate, dissolved in 15% bovine serum albumin, was injected into the calamus scriptorius of the fourth ventricle. The amount and the pattern of labeling of glycerophospholipids synthesized in the motor neurons were determined. Three days after nerve crush there was an accumulation of labeled glycerophospholipids immediately proximal to the injury site. Seven days after crushing, the regenerating nerve incorporated rapidly transported labeled lipids in greater amounts than the contralateral normal nerve; the incorporation was elevated along the entire length of the nerve containing both regenerating axons and the post‐crush sprouting terminals. The difference between the two sides increased up to 14 days, but disappeared as regeneration proceeded (21‐45 days). The “pool” of radioactive lipids remaining in the cell bodies of hypoglossal nuclei, in the segments of nerve, both proximal and distal to the crush site, and in all the segments of uncrushed nerve was similar 6–12 h after labeling. Among the phospholipids, the highest 3H and 14C radioactivity was observed in phos‐phatidylcholine and phosphatidylethanolamine. These results support the hypothesis than an increase in the amount of glycerophospholipids, conveyed by rapid axonal transport, takes place in the first 2 weeks during nerve regeneration. The increased transport of lipids presumably reflects an augmented demand for membrane precursors during the sprouting process.
Increased Acetylcholine Synthesis and Release Following Presynaptic Activity in a Sympathetic Ganglion
Authors: Collier B. , Kwok Y. N. , Welner S. A.
Published: December 2006

Abstract: The acetylcholine (ACh) content of sympathetic ganglia increases above its normal level following a period of preganglionic nerve stimulation. In the present experiments, this extra ACh that accumulates following activity was labeled radioactively from [3H]choline and its specific activity was compared with that of ACh subsequently released during preganglionic nerve stimulation. The specific activity of the released ACh was similar to that of the total tissue ACh, suggesting that the extra ACh mixes fully with endogenous stores. The present experiments also show that transmitter release during neuronal stimulation is necessary for the poststimulation increase in transmitter store. However, the increase was not evident when transmitter release was induced by K+. It is concluded that both transmitter release and impulse invasion of the nerve terminals are necessary for the adaptive phenomenon to manifest itself. The role of choline delivery and choline acetyltransferase activity in generating the poststimulation increase in transmitter store was tested. When choline transport activity measured as choline analogue (homocholine) accumulation increased, ACh synthesis was increased and when transport activity was not increased, neither was ACh synthesis. There was no poststimulation increase in measured choline acetyltransferase activity.
Studies on the Cell‐Free Biosynthesis of CNS Membrane Proteins
Authors: Gilbert Jeffrey M. , Strocchi Paola
Published: December 2006

Abstract: The biosynthesis of CNS membrane proteins was studied in cell‐free systems containing membrane‐bound polysomes (rough endoplasmic reticulum; RER) or free polysomes from rat forebrain. In previous studies of CNS membrane proteins using two‐dimensional gel electrophoretic analysis, five proteins (mol. wt.‐pI: 75K 5.4, 68K 5.6, 61K 5.1, 58K 5.1, and 36K 5.6) were found in ceil membrane fractions including preparations enriched in RER, smooth endoplasmic reticulum, and plasma membranes. One of these proteins, 68K 5.6, was also present in cytosol and comigrated with a microtubule‐associated protein. In our present study, cell‐free systems containing RER were found to synthesize the 75K 5.4, 61K 5.1, and 58K 5.1 proteins. A protein, 34K 5.65, similar (but not identical) to the 36K 5.6 protein was also synthesized. After cell‐free synthesis, the 75K 5.4 and 58K 5.1 proteins could be purified by concanavalin A affinity chromatography. Of the five common membrane proteins previously identified, only the 68K 5.6 protein was synthesized by the free polysome population. The free polysomes were also found to synthesize cyclic AMP binding proteins at 48K and 54K, known from previous studies to be present in both cytosol and plasma membrane fractions in mammalian brain tissue. In conclusion, RER synthesized proteins found exclusively in CNS membrane fractions, whereas free polysomes synthesized those proteins found in both soluble and membrane compartments.