bluuebunny/crossref_metadata_2025 huggingface dataset:
GANGLIOSIDE1−COMPOSITION OF BRAIN IN TAY‐SACHS DISEASE: INCREASED AMOUNTS OF GD2 AND N‐ACETYL‐β‐D‐GALACTOSAMINYL GD1a GANGLIOSIDE
Authors: Iwamori M. , Nagai Y.
Published: October 2006

Abstract— The ganglioside composition of the brain of a patient with Tay‐Sachs disease (TS‐brain) was determined by a newly developed ganglioside‐mapping procedure and compared with that of an age‐matched control brain. GM2 ganglioside was the predominant component in TS‐brain and the following gangliosides were also found, GM1, GD1a, GD1b and GT1 (major gangliosides in normal brain), and GM3, GD3, GD2 and GD1a‐GAN (minor or undetectable components of normal brain). Individual gangliosides were isolated by column chromatography using a combination of DEAE‐Sepharose, Iatrobeads and Silica Gel 60 and their structures were confirmed by comparing them with authentic standards using TLC, analysing their carbohydrate compositions by gas‐liquid chromatography and cleaving them sequentially with glycosidases. The amounts of individual components were measured by quantitative densitometric scanning of the thin‐layer plates. As a reflection of myelin breakdown, no sialosylgalactosyl ceramide was detectable in TS‐brain. Although the total amounts of all gangliosides except GM2 in TS‐brain were low, there were normal molar ratios of the main gangliosides in normal brain, that is, GM1, GD1a, GD1b and GT1. In comparison with the amount of GDla ganglioside, the amounts of GM2, GD2 and GD1a‐GAN, which contain N‐acetylgalactosamine as a terminal carbohydrate residue, were all elevated in TS‐brain. The long chain bases of individual gangliosides contained both C‐18 and C‐20 sphingosine in different ratios and the ratio of C‐20 to C‐18 increased in the gangliosides in the order: GM2 < GM1 < GD1a < GD1a‐GAN < GD1b < GT1 in both normal brain and TS‐brain. In contrast, GD2 and GD3 gangliosides consisted mainly of C‐18 sphingosine. The C‐20 to C‐18 ratios of individual gangliosides in the TS‐brain were lower than those of age‐matched control brain.

Hexosaminidase from Turbo cornutus showed the same specific activity and Km value in catalysing the cleavage of terminal N‐acetylgalactosaminyl residues from GM2, GD2 and GD1a‐GAN, suggesting that the brain gangliosides that increase in Tay‐Sachs disease may be cleaved by the same enzyme.
RAT BRAIN TUBULIN: SUBUNIT HETEROGENEITY AND PHOSPHORYLATION
Authors: Forgue S. Thomas , Dahl June L.
Published: October 2006

Abstract— Evidence for multiple forms of the α and β subunits of tubulin isolated from rat brain has been obtained by means of SDS polyacrylamide gel electrophoresis, isoelectric focusing, and SDS hydroxylapatite column chromatography. Fourteen distinct bands, localized near pH 5.4, were formed when tubulin was subjected to isoelectric focusing in a gradient established with a very narrow range ampholyte mixture. Three tubulin subunits, a1., α2, and β, were separated by sodium dodecyl sulfate polyacrylamide slab gel electrophoresis in a second dimension. The β subunit was more acidic than the α subunits. Brain sections were incubated in tissue culture medium containing 32P1 and radiolabeled tubulin was subsequently isolated and subjected to electrophoresis. Only the β subunit was labeled. All radioactivity was associated with two or three adjacent bands on isoelectric focusing gels.
SOLUBILIZATION AND PARTIAL PURIFICATION OF THE GABA RECEPTOR FROM MOUSE BRAIN AND A BINDING ASSAY FOR THE SOLUBILIZED RECEPTOR1
Authors: Chude Obi
Published: October 2006

Abstract— The GABA receptor from mouse brain was solubilized with lysolecithin. A 56‐fold overall purification and activation were achieved by discontinuous sucrose gradient centrifugation and solubilization. Activation of binding by both procedures was observed. The solubilized receptor has the following binding constants: KD1= 3.5 nM, KD2= 52 nM, Bmax 1= 2.8 pmol/mg protein and Bmax 2= 14 pmol/mg protein for muscimol; KD1= 12 nM, KD2= 470 nM, Bmax 1= 1.4 pmol/mg protein and Bmax 2= 17 pmol/mg protein for GABA. Specific GABA binding was inhibited by imidazoleacetic acid and bicuculline with IC50 values of 250nM and 1 μM respectively. A rapid and sensitive filtration binding assay for the solubilized receptor has been developed. Lysolecithin was also found suitable for the solubilization of acetylcholine receptor from T. californica electroplaques.
DIFFERENTIAL EFFECTS OF AGONAL STATUS ON MEASUREMENTS OF GABA AND GLUTAMATE DECARBOXYLASE IN HUMAN POST‐MORTEM BRAIN TISSUE FROM CONTROL AND HUNTINGTON'S CHOREA SUBJECTS
Authors: Spokes Ernest G. S. , Garrett Nigel J. , Iversen Leslie L.
Published: October 2006

Abstract— GABA and its biosynthetic enzyme glutamic acid decarboxylase (GAD) remained remarkably stable for many hours after death in both human putamen obtained at autopsy and in mouse brain stored under conditions simulating the routine handling of human cadavers. GAD activity was profoundly influenced by agonal status in control but not in choreic subjects. Conversely, GABA concentrations were unaffected by the agonal status but showed a significant age‐related decline. GAD activity and GABA concentrations were positively correlated in sudden death control cases but not in control cases suffering a protracted terminal illness or in choreic subjects. In choreic putamen there was an approximate 50% reduction in GABA concentration and GAD activity (correcting for agonal status) consistent with the hypothesis that striatal GABA‐containing neurones degenerate in this disease. Since GABA concentrations are unaffected by agonal factors they may provide a reliable marker for the integrity of GABA systems provided that control and pathological groups are matched for age and delay in post‐mortem sampling.
THE DISTRIBUTION OF SOLUBLE AND MEMBRANE‐BOUND FORMS OF GLUTAMINASE IN PIG BRAIN
Authors: Nimmo Gillian A. , Tipton Keith F.
Published: October 2006

Abstract— About 10% of the glutaminase activity associated with pig brain mitochondria was readily extractable by a variety of techniques but the remainder was very resistant to extraction. These two forms, which have been termed the soluble and membrane‐bound forms respectively, have been shown to differ in their responses to activation by phosphate and phosphate‐borate containing buffers. Submitochondrial fractionation studies indicated that the soluble form was located in the mitochondrial inner matrix whereas the membrane‐bound form was associated with the inner membrane. The mitochondria associated with the synaptosomes were found to contain only the membrane‐bound form of the enzyme whereas both forms were present in the free brain mitochondria.
Correlations Between Acetylcholinesterase Activity in Guinea‐Pig Iris and Pupillary Function: A Biochemical and Pupillographic Study
Authors: Søli N. E. , Karlsen R. Lund , Opsahl M. , Fonnum F.
Published: October 2006

: Acetylcholinesterase (AChE) in guinea pig iris was inhibited by methylisocyclopentylfluorophosphate (soman) administered topically or parenterally, and enzyme activity was correlated to pupillary diameter by infrared pupillography. After a single topical soman instillation into the conjunctival sac there was an almost linear relationship between the reduction in AChE activity and pupillary diameter. Topical administration of soman at 24‐h intervals in doses capable of almost complete inhibition of AChE in iris was accompanied by a reduced miotic effect of this drug. This was indicated by a reduced rate of the soman‐induced pupillary constriction, a less pronounced reduction in pupillary diameter, and a more rapid return of the pupillary diameter to normal size. The change in pupillary diameter occurred after three daily administrations and remained constant during 31 days of treatment. These observations were seen irrespective of inhibition of blood AChE. The decrease in response to repeated administration could not be explained by a reduced inhibitory effect of soman on AChE, by a more rapid de novo synthesis of AChE, or by a change in the number of the muscarinic receptors as determined by quinuclidinyl benzilate binding. When soman or DFP was administered subcutaneously in high doses a severe AChE inhibition was obtained in iris without any concomitant miosis.
Physical and Kinetic Properties of Choline Acetyl Transferase from Rat and Bovine Brain
Authors: Ryan Rick , McClure William O.
Published: October 2006

Several physical and kinetic properties of highly purified choline acetyltransferase (EC 2.3.1.6., Acetyl‐CoA:choline O‐acetyltransferase) from rat and bovine brain were investigated. The molecular weight of the enzyme from rat cerebrum was 65,000 measured by gel filtration and 66,000 measured by acrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Two species of the enzyme detected in bovine caudate nuclei were studied using the same procedures. The major transferase species, bovine I (Bov I), yielded values of molecular weights of 69,000 and 70,000, respectively. The minor bovine acetylase species (Bov II) had a molecular weight of 67,000 daltons by gel filtration. The dependence of reaction rates catalyzed by Bov I and rat choline acetyltransferase on temperature and pH was examined. The two enzymes possess apparent activation energies of 21.0 and 20.0 kcal/mol, respectively. Both displayed a rather broad pH optimum, extending into the alkaline region, and were activated by either NaCl or KCl. Neither enzyme was activated or inhibited by a wide range of concentrations of acetyl‐CoA or choline. Initial velocity studies for both enzymes indicated a sequential mechanism, in which both substrates must bind before either product is released. For Bov I, the Michaelis constants for acetyl‐CoA and choline were 16.5 μM and 0.714 mm, respectively; the equivalent constants for rat CAT were 46.5 μM and 1.0 mm. Product inhibition studies with the rat enzyme showed that acetylcholine was competitive with respect to choline, and noncompetitive with respect to acetyl‐CoA, while CoA inhibited competitively with respect to acetyl‐CoA and noncompetitively with respect to choline. The values of K1, obtained for acetylcholine and CoA were 45.5 mm and 37.7 μM, respectively.
Hippocampal Free Amino Acids in Alzheimer's Disease
Authors: Tarbit I. , Perry E. K. , Perry R. H. , Blessed G. , Tomlinson B. E.
Published: October 2006

The free amino acid content of the hippocampus, obtained at postmortem, has been analysed in cases of Alzheimer's disease and compared with normal cases. There were no significant differences in the levels of 23 amino acids including the transmitter candidates γ‐aminobutyric, glutamic or aspartic acids. This finding is interpreted in relation to present knowledge of transmitter pathways in the region of the hippocampus. A tendency for some amino acids to be increased in the Alzheimer group reached statistical significance for arginine. This observation is consistent with increased proteolytic or peptidase activity in Alzheimer's disease.
Storage and Release of Noradrenaline in Hypothalamic Synaptosomes
Authors: West D. P. , Fillenz M.
Published: October 2006

Abstract: The noradrenaline storage capacity of vesicles in hypothalamic synaptosomes was measured by incubating them with [3H]noradrenaline under saturating conditions. The normal noradrenaline content is 52% of storage capacity. Incubation or superfusion with 50 mm‐potassium causes calcium‐dependent release from the vesicles. Such release reduces not only the vesicular content, but also the noradrenaline storage capacity. This suggests that after exocytosis vesicles cannot refill with noradrenaline.
Allosteric Properties of Ox Brain Nicotinamide–Adenine Dinucleotide Dependent Isocitrate Dehydrogenase
Authors: Willson Vivian J. C. , Tipton Keith F.
Published: October 2006

Abstract: The kinetic and regulatory properties of a partly purified preparation of ox brain NAD+‐dependent isocitrate dehydrogenase have been studied at pH 7.5. The enzyme exhibits rate cooperativity with respect to isocitrate but shows normal hyperbolic kinetics with respect to NAD+. ADP activates the enzyme by decreasing the substrate concentrations that are necessary to give half‐maximal velocity, but it has no effect on the Hill constant for isocitrate unless Mg2+ ions are replaced by Mn2+ ions in the reaction mixture. Citrate and tricarballylate activate the enzyme in a similar fashion to ADP. Higher concentrations of citrate cause inhibition but this could be overcome by raising the concentration of Mg2+ ions, suggesting that the inhibition by this compound might be due to its acting as a chelating agent. NADH and NADPH were competitive inhibitors with respect to NAD+ but the product, 2‐oxoglutarate, was not inhibitory. γ‐Aminobutyrate and a number of other compounds involved in the γ‐aminobutyrate pathway had no significant effect on the activity of the enzyme.
Nuclear DNA Ligase and Its Action on Chromatin DNA in Neuronal, Glial and Liver Nuclei Isolated from Adult Guinea Pigs
Authors: Inoue Nobuo , Kato Takahiko
Published: October 2006

Abstract: DNA ligase activities were measured in neuron‐rich and glial nuclear preparations and liver nuclei isolated from adult guinea pigs. The enzymatic properties of cerebral and liver nuclear DNA ligases were studied with isolated nuclei and nuclear extracts. ATP (Km= 46–48 μM) and bivalent cation (Mg2+ or Mn2+) were required for the maximal activities in cerebral and liver nuclei. β‐Mercaptoethanol did not affect the activities, but N‐ethylmaleimide and p‐chloromercuribenzoate completely inhibited the activities. Deoxyadenosine‐5′‐triphosphate partially inhibited the activities in both cerebral and liver nuclei. An interdependent effect of Na+ and Mg2+ on the enzyme activities was observed. A high concentration (200 mM) of Na+ activated both enzymes and shifted to the acid side the optimal pH for both enzymes. DNA ligase was more easily extracted with lower concentrations of NaCl from liver nuclei than from cerebral nuclei, but the extraction curves from both nuclear species reached a plateau level (92% of total activities of nuclear enzymes) at 200 mM‐NaCl. Apparent Km for the substrate [32P]phosphoryl DNA was determined according to a modification of the Michaelis‐Menten equation, which was applied for the case where an unknown amount of substrate nicks in chromatin DNA coexisted with the nicks in exogenous substrate DNA. Neuronal and glial nuclear enzymes had similar Km values (about 20 μg of [32P]phosphoryl DNA/ml), but the liver nuclear enzyme had a higher Km value (54 μg of [32P]phosphoryl DNA/ml). The modified Michaelis‐Menten equation provided the amounts of nicks available as substrate in chromatin DNA of isolated nuclei. Neuronal and glial nuclei contained 1.5 and 0.29 pmol of nicks/μg of nuclear DNA, respectively, in contrast to an intermediate amount of nicks in liver nuclei (0.63 pmol/μg of nuclear DNA). DNA ligase activity in neuronal nuclei [312 units (fmol of 5′‐phosphomonoester converted into a phosphatase‐resistant form per min at 37°C) per μg of nuclear DNA] was 11‐fold higher than that in glial nuclei [28.7 units/μg of nuclear DNA]. Liver nuclei contained an intermediate activity [54.7 units/μg of nuclear DNA].
Conversion of 3,4‐Dihydroxyphenylalanine and Deuterated 3,4‐Dihydroxyphenylalanine to Alcoholic Metabolites of Catecholamines in Rat Brain
Authors: Edwards David J. , Rizk Marguerite
Published: October 2006

Abstract: We have investigated the effects of 3,4‐dihydroxyphenylalanine l‐DOPA) and its deuterated analogue on the concentrations of alcoholic metabolites of catecholamines in rat brain by means of gas chromatography/mass spectrometry with selected‐ion monitoring. Whole brain concentrations of the two neutral norepinephrine metabolites, 3‐methoxy‐4‐hydroxyphenylethylene‐glycol (MHPG) and 3,4‐dihydroxyphenylethyleneglycol (DHPG), were significantly increased in a dose‐dependent manner by a single intraperitoneal injection of l‐DOPA. Both MHPG and DHPG, as well as the corresponding dopamine metabolites, reached a maximum 1 h after injection. Brain MHPG and DHPG concentrations were elevated by 78 and 134%, respectively, 1 h after injection of 150 mg/kg l‐DOPA. Analyses of discrete brain regions revealed that concentrations of the norepinephrine metabolites were elevated uniformly in all regions, except that MHPG showed a greater increase in the cerebellum than in other regions. The latter result appeared to be explained by the finding that 52% of the total MHPG in the cerebellum was unconjugated (compared to 15% in the whole brain). l‐DOPA caused a proportionately greater increase in free MHPG than in total MHPG in the cerebellum and brain stem. By using deuterated l‐DOPA in place of l‐DOPA and measuring both the deuterated and nondeuterated norepinephrine metabolites, we demonstrated that virtually all of the increases in MHPG and DHPG were due to the conversion of the exogenous l‐DOPA to norepinephrine. Thus, the effects of norepinephrine metabolism need to be considered in attempts to understand clinical and behavioral effects of l‐DOPA.
Effect of Hypo‐ and Hyperthyroidism on Hexokinase in the Developing Cerebellum of the Rat
Authors: Gutekunst Diane I. , Wilson John E.
Published: October 2006

Abstract: Total hexokinase levels (units/g tissue) have been measured during postnatal development of the cerebellum in control, hypothyroid, and hyperthyroid rats. In addition, distribution of hexokinase in the developing cerebellum has been observed with an immunofluorescence method. Hypothyroidism delays the normally observed postnatal increase in total hexokinase activity, whereas hyperthyroidism accelerates the increase. In normal animals, hexokinase levels in maturing Purkinje cells pass through a transient increase, with maximal levels at approximately 8 days postnatally followed by rapid decline to relatively low levels by 12 days; hypothyroidism delays this transient increase and subsequent decline, but hyperthyroidism does not appear to affect markedly the timing of this phenomenon. Cerebellar glomeruli are relatively enriched in hexokinase content, as judged by their intense fluorescence. Hypothyroidism delays the development of intensely stained glomeruli. Hyperthyroidism did not appear to cause precocious increase in numbers of glomeruli but may have increased the rate at which the hexokinase was assimilated by newly formed glomeruli. The effects of hypo‐ and hyperthyroidism on total cerebellar hexokinase levels are interpreted in terms of the effect of thyroid hormone on the biochemical maturation of synaptic structures rich in hexokinase.
Identification of Strychnine Binding Sites in the Rat Retina
Authors: Schaeffer James M , Anderson Susanne M
Published: October 2006

Abstract: [3H]Strychnine specifically binds to membrane fractions isolated from rat retinae. The binding is saturable, with an apparent dissociation constant, KD, of 14.3 × 10−9 M and 205 fmol bound/mg protein. Specific binding is time‐dependent and proportional to protein concentration. Glycine and taurine are equally potent inhibitors of [3H]strychnine binding (Ki= 4 × 10−5 M); no other amino acids endogenously present in the retina inhibited [3H]strychnine binding.
Quinuclidinyl Benzilate Binding in House Fly Heads and Rat Brain
Authors: Jones Stephen W. , Sumikawa Katumi
Published: October 2006

Abstract: House fly heads contain a binding site for 3‐quinuclidinyl benzilate (QNB) that is quite similar in pharmacology to the muscarinic acetylcholine receptor of vertebrate tissues. The house fly site binds [3H]QNB reversibly with a Kd of 260 PM and Bmax of 1 pmol/g of heads from direct binding measurements. The Kd calculated from the ratio of the dissociation rate constant (2 × 10−4 sec−1) to the association rate constant (2.5 × 106 M−1 Sec−1) was 80 pM. The house fly site binds (‐)quinuclidinyl benzilate preferentially, as do classic muscarinic receptors. The binding is also sensitive to other muscarinic antagonists and agonists. Nicotinic and other drugs are no more effective on the house fly site than they are on the rat brain muscarinic receptor itself. These binding studies suggest that the house fly QNB binding site is a muscarinic receptor.
Bovine Adrenal Tyrosine Hydroxylase: Comparative Study of Native and Proteolyzed Enzyme, and Their Interaction with Anions
Authors: Vigny Annette , Henry Jean‐Pierre
Published: October 2006

Abstract: The pH optimum of native adrenal medulla tyrosine hydroxylase activity is shifted from 5.8 to 6.4 by polyanions (heparin, dextran sulphate), salts (NaCl, Na2SO4) and the anionic buffer 2‐(N‐morpholino)ethanesulphonic acid (MES). Simultaneously, the activity at the optimal pH is increased. Kinetic studies have shown that this activation is associated with a decrease of the apparent Km of the enzyme for the cofactor 6,7‐dimethyltetrahydropterin (DMPH4) and an increase in the Vmax for tyrosine and DMPH4. The Km for the tyrosine remained unchanged. These data have been interpreted in terms of the polyelectrolyte theory. The adsorption of tyrosine hydroxylase on various affinity gels containing heparin, dextran sulphate or unsulphated polymer dextran as ligands indicate that the activation of the enzyme is mediated by electrostatic interactions with the anionic species. The site of electrostatic interaction possesses some specificity since the binding constants are higher for heparin or dextran sulphate than for NaCl or MES buffer. Moreover, 3‐(N‐morpholino)propanesulphonic acid (MOPS) a slightly structurally different buffer inhibits the enzyme activity whereas N‐(2‐acetamido)‐2‐amino‐ethanesulphonic acid (ACES) has no effect. A limited proteolytic digestion which preserves the enzymatic activity, destroys the effects of the anions. The isoelectric point and the molecular parameters of tyrosine hydroxylase are markedly altered after limited digestion. It is therefore suggested that the interaction between the hydroxylase and anionic compounds occurs on a part of the protein which is different from the active site and which is lost by proteolysis. This portion of the protein might be involved in regulation of native tyrosine hydroxylase.
Cysteinesulfinic Acid Decarboxylase Activity in the Mammalian Nervous System: Absence from Axons
Authors: Sturman J. A.
Published: October 2006

The activity of cysteinesulfinic acid decarboxylase (CSAD, EC 4.1.1.29) in extracts of liver of seven mammals varied greatly, whereas in extracts of brain from the same species, the variation was less marked. CSAD activity was readily measured in extracts of spinal cord from the same species, except those from rhesus monkey and man. The most noteworthy observation was the complete absence of CSAD activity in extracts of optic nerves and of sciatic nerves from all seven mammals. This suggests that taurine biosynthesis does not occur within axons and that intraaxonal taurine is supplied by axonal transport from the cell body.
Effect of Cyclic AMP‐Dependent Protein Phosphorylating Conditions on the pH‐Dependent Activity of Tyrosine Hydroxylase from Beef and Rat Striata
Authors: Pollock Robert J. , Kapatos Gregory , Kaufman Seymour
Published: October 2006

Abstract Tyrosine hydroxylase (TH, EC 1.14.16.2) from beef brain striata was purified 23‐fold from an extract of an acetone powder. If this enzyme preparation is treated with a cyclic AMP‐dependent protein phosphorylation system, there is a change in the pH dependence of the enzyme activity. The pH optimum at saturating tetrahydrobiopterin (BH4) concentration is shifted from below pH 6 to about pH 6.7. At pH 7, activation is expressed mainly as an increase in Vmax, whereas at pH 6, activation is expressed mainly as a decrease in Km for the pterin cofactor. Further, even with the control enzyme the Km for pterin cofactor declines precipitously as the pH is increased from 6 toward neutrality. Similar data were obtained with G‐25 Sephadex‐treated rat striatal TH. Experiments in which rat striatal synaptosomes were used demonstrated that the in situ activation of TH by phosphorylating conditions is expressed primarily as an increase in the maximum rate of dopamine synthesis. These results indicate that changes in TH activity caused by cyclic AMP‐dependent protein phosphorylation will depend to a large extent on the pH of the TH environment.
Aging Decreases Oxidative Metabolism and the Release and Synthesis of Acetylcholine
Authors: Gibson Gary E. , Peterson Christine
Published: October 2006

Acetylcholine (ACh) synthesis in vivo is known to decrease during the aging process (senescence). To elucidate the molecular mechanism(s) of this age‐related decline, we studied brain slices from 3‐, 10‐, and 30‐month‐old mice of two strains (C57B1 and Balb/c). In low K+ media, oxidative metabolism as measured by 14CO2 production decreased with aging from 100% (3 months) to 85% (10 months) or 71% (30 months) whether [U−14C]glucose, [3,4‐14C]glucose, or [l‐14C]pyruvate was the substrate. In the aged brain (3 months) the increase in 14CO2 production with K+ stimulation was about twofold higher than in the young brain (3 months). Thus, in high K+ media, only slight decreases (<10%) in oxidative metabolism occurred with aging. Changes in ACh synthesis paralleled the decreases in 14CO2 production. Synthesis of [14C]ACh from [U‐14C]glucose in low K+ media declined from 100% (3 months) to 85% (10 months) or 66% (30 months), while in high K+ media only slight decreases (<10.5%) occurred with aging. The Ca2+‐dependent, K+‐stimulated release of [14C]ACh declined from 100% (3 months) to 58% (10 months) or 25% (30 months). Only the decrease in the release of ACh declined to the same extent as the reduced in vivo synthesis of ACh with aging. The results suggest that decreases in oxidative metabolism, ACh synthesis, and in the release of ACh contribute to a reduction in cholinergic function in the senescent brain.
Neonatal Hormone Treatment and Maturation of the Pineal Noradrenergic System: Hydrocortisone and Thyroxine
Authors: Yuwiler A. , Brammer Gary L.
Published: October 2006

Abstract The circadian release of norepinephrine from nerve terminals in the pineal gland drives acetyl‐CoA:serotonin N‐acetyltransferase (NAT; EC 2.3.1.5) activity in the adult pineal from a daytime low to a nighttime high. In the newborn, enzyme activity is intermediate between the adult's daily extremes and has only a small circadian fluctuation. With age, these fluctuations increase in amplitude until the adult pattern is attained at about days 10–12. Treatment of neonates with thyroxine for the first 3 days of life accelerated, whereas administration of hydrocortisone acetate at birth retarded the developmental decline in daytime serotonin‐N‐acetyltransferase activity. Maximal differences in daytime enzyme activity of controls and thyroxine‐treated animals were seen at day 4 and between controls and steroid‐treated pups at day 8. Desipramine treatment increased NAT activity in 8‐day‐old animals; hydrocortisone‐treated animals were least affected. Freshly cultured pineals from steroid‐treated animals were more responsive to low, and less responsive to high, concentrations of norepinephrine than glands from thyroxine‐treated or control animals. They were also less responsive to isoproterenol both in acute and 48‐h organ culture. Pineals from hydrocortisone‐treated animals in culture accumulated less exogeneous norepinephrine than glands from controls but released a greater fraction of their content on transfer to fresh medium. Normal and steroid‐treated animals released the same fraction of their norepinephrine contents into the medium when reuptake was blocked by desipramine (DMI).
Effects of Cations on (Ca2++ Mg2+)‐Activated ATPase from Rat Brain
Authors: Robinson Joseph D.
Published: October 2006

Abstract: With a partially purified, membrane‐bound (Ca + Mg)‐activated ATPase preparation from rat brain, the K0.5 for activation by Ca2+ was 0.8 p μm in the presence of 3 mm‐ATP, 6 mm‐MgCl2, 100 mM‐KCI, and a calcium EGTA buffer system. Optimal ATPase activity under these circumstances was with 6‐100 μm‐Ca2+, but marked inhibition occurred at higher concentrations. Free Mg2+ increased ATPase activity, with an estimated K0.5, in the presence of 100 μm‐CaCl2, of 2.5 mm; raising the MgCl2 concentration diminished the inhibition due to millimolar concentrations of CaCl2, but antagonized activation by submicromolar concentrations of Ca2+. Dimethylsulfoxide (10%, v/v) had no effect on the K0.5 for activation by Ca2+, but decreased activation by free Mg2+ and increased the inhibition by millimolar CaCl2. The monovalent cations K+, Na+, and TI+ stimulated ATPase activity; for K+ the K0.5 was 8 mm, which was increased to 15 mm in the presence of dimethylsulfoxide. KCI did not affect the apparent affinity for Ca2+ as either activator or inhibitor. The preparation can be phosphorylated at 0°C by [γ‐32P]‐ATP; on subsequent addition of a large excess of unlabeled ATP the calcium dependent level of phosphorylation declined, with a first‐order rate constant of 0.12 s−1. Adding 10 mm‐KCI with the unlabeled ATP increased the rate constant to 0.20 s−1, whereas adding 10 mm‐NaCl did not affect it measurably. On the other hand, adding dimethyl‐sulfoxide slowed the rate of loss, the constant decreasing to 0.06 s−1. Orthovanadate was a potent inhibitor of this enzyme, and inhibition with 1 μm‐vanadate was increased by both KCI and dimethylsulfoxide. Properties of the enzyme are thus reminiscent of the plasma membrane (Na + K)‐ATPase and the sarcoplasmic reticulum (Ca + Mg)‐ATPase, most notably in the K+ stimulation of both dephosphorylation and inhibition by vanadate.
Glutamate Decarboxylase Distribution in Discrete Motor Nuclei in the Cat Brain
Authors: Nieoullon André , Dusticier Nicole
Published: October 2006

Abstract: The distribution of activity of glutamate decarboxylase (GAD), the enzyme synthesising γ‐aminobutyric acid (GABA), was measured in the cat brain by means of microdissection of the structures from frozen slices and a radioisotopic assay for the enzyme. About 20 cerebral regions were chosen for study because of their role in sensorimotor integration. GAD presented an uneven distribution among these areas. Highest activities were found in the basal ganglia, particularly in the substantia nigra and in the globus pallidus, and to a lesser extent in the cerebellum. Relatively low levels of the enzyme were found in the thalamus and in the cerebral motor cortex. Special detailed studies were made in the caudate nucleus, the substantia nigra, and in the red nucleus for the purpose of defining the intranuclear distribution of their GABAergic innervation. There were only small differences in the rostro‐caudal distribution of the enzyme in the head of the caudate nucleus but GAD activity was higher in the ventral than in the dorsal part of the structure. In the substantia nigra, GAD activity was high in both the medial and intermediate thirds of the structure. The GAD activity decreased from the caudal to the rostral part of the nucleus. GAD levels were lower in the caudal part of the red nucleus than in the rostral part. These results indicate that GABA would be present as a putative neurotransmitter in many motor nuclei of the cat brain. In view of the general inhibitory action of this amino acid, this could be related to the presence of inhibitory responses widely distributed in these nuclei as identified by mean of electrophysiological studies. The origin of these GABAergic innervations in many cases remains to be determined.
L‐Aspartate Transport into Plasma Membrane Vesicles Derived from Rat Brain Synaptosomes
Authors: Marvizón J. G. , Mayor F. , Aragón M. C. , Giménez C. , Valdivieso F.
Published: October 2006

Abstract: Aspartate uptake by membrane vesicles derived from rat brain was investigated. The uptake is dependent on a Na+ gradient ([Na+] outside > [Na+] inside). Active transport of aspartate is strictly dependent upon the presence of sodium and maximal extent of transport is reached when both Na+ and Cl− ions are present. The uptake is transport into an osmotically active space and not a binding artifact as indicated by the effect of increasing the medium osmolarity. The uptake of aspartate is stimulated by a membrane potential (negative inside), as demonstrated by the effect of the ionophore carbonyl cyanide m‐chlorophenylhydrazone and anions with different permeabilities. The presence of ouabain, an inhibitor of (Na++ K+)‐ATPase, does not affect aspartate transport. The kinetic analysis shows that aspartate is accumulated by two systems with different affinities, showing Km and Vmax values of similar order to those found in slightly “cruder“ preparations. Inhibition of the l‐aspartate uptake by d‐aspartate and d‐ and l‐glutamate indicates that a common carrier is involved in the process, this being stereospecific for the d‐ and l‐glutamate stereoisomers.
A Comparison of In Vitro‐ and In Vivo Phosphorylated Neurofilament Polypeptides
Authors: Julien Jean‐Pierre , Mushynski Walter E.
Published: October 2006

Abstract: Neurofilament polypeptides phosphorylated in vitro by incubation of neurofilament‐enriched preparations from rat CNS with [γ‐32P]ATP were compared with the corresponding polypeptides labeled in vivo by injection of 32Pi into the lateral ventricles of rats. Autoradiography of sodium dodecyl sulfate (SDS)‐polyacrylamide gels revealed that the major phosphorylated species in both preparations were the three neurofilament subunits, which have molecular weights of 200K, 145K, and 68K. However, the relative levels of 32P detected in the three in vitro‐labeled subunits differed from the relative in vivo levels. The two larger neurofilament polypeptides displayed similar 32P isoprotein distribution patterns on two‐dimensional gels, whereas additional isoproteins were seen in the in vitro‐labeled 68K species. Limited proteolysis in SDS‐polyacrylamide gels revealed the presence of common phosphopeptides in the corresponding pairs of in vitro‐ and in vivo‐labeled subunits, but the in vivo‐labeled 145K and in vitro‐labeled 200K polypeptides contained additional digestion products. Two‐dimensional peptide mapping of the 68K polypeptide digested with a mixture of trypsin and chymotrypsin indicated that this component was phosphorylated at a single, identical site, both in vivo and in vitro. These results indicate that the protein kinase that copurifies with neurofilament preparations may be involved in their in vivo phosphorylation.
Heterogeneity of the S‐100 Protein Specific Binding Sites in Synaptosomal Particulate Fractions and Subfractions
Authors: Donato Rosario
Published: October 2006

Abstract: The specific interaction of S‐100 protein with synaptosomal particulate fractions (SYN) was further investigated with special reference to the number of binding components and their localization in synaptosomal subfractions. Binding studies were conducted on SYN from various CNS regions, on synaptosomal subfractions from the cerebral cortex, and on cerebral cortex SYN under Various conditions. The results suggest that S‐100 binds to two populations of membrane sites: high‐affinity sites, which seem to be confined to neuronal membranes (synaptosomal plasma membranes and synaptic vesicles), and low‐affinity sites, which are also detected in other membranes. The data are consistent with the view that the biphasic profile of S‐100 binding to SYN does not result from heterogeneity of the S‐100 molecule, and that the Ca2+ conformation of the protein is as important as the proper conformation of the binding site for full expression of high‐affinity binding.
The Specific Interaction of S‐100 Protein with Synaptosomal Particulate Fractions. Modulation of Binding by Fractional Occupancy of Sites and by the Physical State of Membranes
Authors: Donato Rosario
Published: October 2006

Abstract: The nonlinearity of single components of the Scatchard plot of S‐100 binding to synaptosomal particulate fractions (SYN) and the observation that dilution of the 125I‐labeled S‐100 site complex results in a greater extent of dissociation of the tracer in the presence than in the absence of an excess of unlabeled S‐100 suggest that sites change their binding behavior depending on fractional occupancy. To study this aspect of the interaction in more detail, 125I‐labeled S‐100 binding experiments were conducted in the presence of, or after preincubation of SYN with various concentrations of, unlabeled S‐100. The results indicate that: (a) S‐100 synaptosomal sites do change their binding behavior depending on fractional occupancy; and (b) the nonrapid equilibrium between bound S‐100 and the medium, which has been referred to as the formation of a tight complex between S‐100 and its binding sites, is related to the activation of high‐affinity sites. However, no univocal interpretation of these data in terms of binding model can be offered at present, as the binding models currently employed in the analysis of ligand‐site interactions can each account for only part of the results described in this report. In any case, data obtained by studying 125I‐labeled S‐100 binding to untreated SYN at 2°C and to prefixed SYN at 37°C indicate that the physical state of membranes influences both the extent of the interaction and the binding behavior of the sites.
The Relationship of Myelin Basic Protein (Arginine) Methyltransferase to Myelination in Mouse Spinal Cord
Authors: Crang A. J. , Jacobson W.
Published: October 2006

Abstract: The relationship between the activity of myelin basic protein (arginine) methyltransferase and myelination in the mouse spinal cord has been examined. The activity of this methylase increases between 8 and 45 days postnatal age and correlates well with other parameters of myelination. A comparison of myelin basic protein methylase with histone methylase activity during development indicates that each is a distinct, specific enzyme activity. Together, these results are considered to establish myelin basic protein methylase as a myelination‐related enzyme.
Postnatal Development of the Light Response of the Dopaminergic Neurons in the Rat Retina
Authors: Morgan William W. , Kamp Cylia W.
Published: October 2006

Abstract: The effect of light on retinal dopamine (DA) synthesis and content in dark‐adapted rats was assessed 15 h and 2, 4, 7, and 16 days after eye opening (13 to 14 days after birth). The accumulation of dihy‐droxyphenylalanine (DOPA) following inhibition of its decarboxylation was used to estimate DA synthesis. At 7 and 16 days, but not earlier, light significantly augmented DOPA formation. These increases were as dramatic as those reported for adult rats. DA in dark‐adapted retinas ranged from 0 (undetectable) at 15 h to 83% of adult levels at 16 days, but were only 36% of that of adult retinas at 7 days. Light produced a significant decline in DA at 16 days but not at any other time point. These results indicate that the dopaminergic neurons synthesize transmitter and respond to light at a time when the neuronal pools of DA are not yet mature.
Subcellular Distribution of Human Brain Aldehyde Dehydrogenase
Authors: Inoue Kazuyoshi , Lindros Kai O.
Published: October 2006

Abstract: Two human brain surgery biopsies and one autopsy sample were subjected to subcellular fractionation. With either 0.12 or 6 mM‐acetaldehyde as substrate, about half of the total aldehyde dehydrogenase activity was found in the mitochondrial (+ synaptosomal) fraction and less activity in the cytosolic, nuclear, and microsomal fractions. High‐affinity activity was found only in the mitochondrial fraction. The enzyme in all fractions had a higher affinity for indole‐3‐acetaldehyde than for acetaldehyde. The kinetic data indicate the presence of several distinct aldehyde dehydrogenase isozymes that have ample capacity to oxidize both aliphatic and aromatic aldehydes in human brain.
Interrelationships of Ubiquinone and Sterol Syntheses in Cultured Cells of Neural Origin
Authors: Volpe Joseph J. , Obert Kathleen A.
Published: October 2006

Abstract: Ubiquinone synthesis has been studied in cultured C‐6 glial and neuroblastoma cells by utilizing an inhibitor, 3‐β‐(2‐diethylaminoethoxy) androst‐5‐en‐17‐one hydrochloride (U18666A), of cholesterol biosynthesis. Exposure of C‐6 glial cells to nanomolar quantities of U18666A caused a marked inhibition of total sterol synthesis from [14C]acetate or [3H]mevalonate within minutes. A 95% inhibition was apparent after a 3‐h exposure to 200 ng/ml of U18666A. These observations, together with studies of the incorporation of radioactivity from the two precursors into cholesterol, desmosterol, lanosterol, and squalene, indicated that although the most sensitive site to inhibition by U18666A is desmosterol reduction to cholesterol, a major site of inhibition is demonstrable at a more proximal site, perhaps squalene synthetase. As a consequence of the latter inhibition, exposure of C‐6 glial cells to U18666A caused a marked stimulation of incorporation of [14C]acetate or [3H]mevalonate into ubiquinone. Over a wide range of U18666A concentrations, the increase in ubiquinone synthesis was accompanied by an approximately similar decrease in total sterol synthesis. Whereas in the absence of U18666A only approximately 7% of the radioactivity incorporated from [3H]mevalonate into isoprenoid compounds was found in ubiquinone, in the presence of the drug approximately 90% of incorporated radioactivity was found in ubiquinone. The reciprocal effects of U18666A on ubiquinone and sterol syntheses were apparent also in the neuronal cells. The data thus demonstrate a tight relationship between ubiquinone and sterol biosyntheses in cultured cells of neural origin. In such cells ubiquinone synthesis is exquisitely sensitive to the availability of isoprenoid precursors derived from the cholesterol biosynthetic pathway.
Fluorescence Studies of Red Blood Cell Membranes from Individuals with Huntington's Disease
Authors: Sumbilla Carlota , Lakowicz Joseph R.
Published: October 2006

Abstract: Fluorescence spectroscopic methods were used to investigate and compare the properties of erythrocyte membranes from individuals with Huntington's Disease (HD) and from normal individuals. Erythrocyte ghosts were labeled with four different fluorescent probes: 1,6‐diphenylhexatriene (DPH); 6‐lauroyl‐2‐(dimethylamino)‐naphthalene (Laurdan); 2‐(4‐maleimide anilino)‐naphthalene‐6‐sulfonic acid (MIANS) and 5‐(iodoacetamidoethyl)aminonaphthalene‐1‐sulfonic acid (IAEDANS). DPH is sensitive to the microviscosity of the membranes. Laurdan is highly sensitive to the polarity and relaxation time of its environment. MIANS and IAEDANS both react covalently with sulfhydryl groups in membrane proteins. Erythrocyte membranes were labeled separately with each of these four probes, and we measured the centers of gravity of the fluorescence emission, the fluorescence anisotropies, and the fluorescence lifetimes. In 10 separate experiments, including a total of 24 patients and 14 control individuals, we found no significant differences between the two groups for any of the probes or spectral parameters. These results do not support the existence of a “generalized membrane defect” in individuals with HD.
Biochemical Evidence for Alteration of Neostriatal Dopaminergic Function by 5,7‐Dihydroxytryptamine
Authors: Liston Dane R. , Franz Donald N. , Gibb James W.
Published: October 2006

Abstract: Unilateral injection of 5,7‐dihydroxytryptamine (DHT) into the rat neostriatum markedly reduced not only striatal tryptophan hydroxylase (TPH) activity but also striatal tyrosine hydroxylase (TH) activity and dopamine (DA) concentration measured 10‐15 days later. The decrease in striatal TH activity was dose related over the range of 8‐32 pg of DHT; a dose of 16 pg reduced striatal TH activity to 40‐50% of control, DA concentration to 38% of control, and TPH activity to 5520% of control. Intrastriatal injection of 16 pg of DHT reduced TH activity in the ipsilateral substantia nigra to 51% of control. Pretreatment with amfonelic acid, a potent DA uptake inhibitor, significantly reduced the effect of DHT on striatal and nigral TH activity and striatal DA concentration without affecting the DHT‐induced decrease in striatal TPH activity. Desmethylimipramine (5 and 25 mglkg) had no effect on the DHT‐induced decrease in striatal TH activity. Striatal choline acetyltransferase and glutamic acid decarboxylase activities were not decreased by 16 pg of DHT. The results indicate that DHT can alter dopaminergic function in the rat neostriatum through a direct effect of the drug on DA neurons.
Subcellular Localization in Rat Brain of Angiotensin I‐Generating Endopeptidase Activity Distinct from Cathepsin D
Authors: Tonnaer Jeroen A. D. M. , Wiegant Victor M. , de Jong Wybren
Published: October 2006

Abstract: The generation of angiotensin I from the artificial renin substrate tetradecapeptide by proteolytic enzymes in rat brain tissue was studied. The involvement of endopeptidase activity in the enzymatical cleavage of the renin substrate was inferred from the simultaneous accumulation of both angiotensin I and the complementary tetrapeptide Leu‐Val‐Tyr‐Ser on incubation of tetradecapeptide with rat brain tissue. This endopeptidase activity was active over a pH range of 3.5–7.5. In contrast, cathepsin D released angiotensin I from tetradecapeptide only at acidic pH. The angiotensin I accumulation on incubation of tetradecapeptide with brain endopeptidase activity was only partly inhibited in the presence of an excess of the carboxyl protease inhibitor N‐acetyl pepstatin. Further, the brain endopeptidase activity displayed a subcellular localization different from that of acid protease activity. It is concluded that angiotensin I can be generated in the brain by soluble endopeptidases, which are distinct from cathepsin D.
Capsaicin Does Not Change Tissue Levels of Glutamic Acid, Its Uptake, or Release in the Rat Spinal Cord
Authors: Singer Ernst A. , Sperk Gunther , Schmid Rainer
Published: October 2006

Abstract: Capsaicin treatment (50 mglkg, subcutaneous) of newborn rats resulted in a 75% decrease of substance P immunoreactivity in the dorsal spinal cord of the adult animal, but failed to affect levels of the proposed sensory neurotransmitter glutamic acid or to alter high‐affinity uptake of [3H]glutamic acid into synaptosomes of the same tissue. Furthermore, capsaicin (30 μM) in vitro had no influence on the release of [3H]glutamic acid from spinal cord P2 fractions of untreated adult rats, but induced a marked release of substance P. The results suggest that, in contrast to substance P fibers, neurons containing glutamic acid are not sensitive to capsaicin. Eleven other neurochemical parameters measured in the spinal cord did not appear to be changed by the treatment with capsaicin, suggesting a considerable neurochemical selectivity of the lesion.
Early Stimulation of Phosphatidylcholine Biosynthesis During Wallerian Degeneration of Rat Sciatic Nerve
Authors: Natarajan V. , Yao Jeffrey K. , Dyck Peter J. , Schmid Harald H. O.
Published: October 2006

Abstract: Phospholipid metabolism was studied in rat sciatic nerve during Wallerian degeneration induced by crush injury. Portions of crushed sciatic nerve, incubated with labeled substrates, showed significantly higher phosphatidylcholine synthesis than normal nerve, prior to any measurable alterations of phospholipid composition. Maximum synthesis occurred 3 days after crush injury, at which time the metabolism of other phospholipids was unchanged. After a rapid decrease in biosynthetic activity, a second phase of enhanced phosphatidylcholine synthesis occurred, beginning 6 days after crush injury. Increased incorporation of [33P]phosphate, [2‐3H]glycerol, and [Me‐14C]‐ choline indicated stimulation of de novo synthesis of phosphatidylcholine 3 days after injury. Neither base exchange reactions nor sequential methylation of ethanolamine phospholipids contributed significantly to phosphatidylcholine synthesis. Assay of certain key enzymes under optimal conditions in subcellular fractions of sciatic nerve revealed higher activities of cholinephosphate cytidyltransferase, choline phosphotransferase, and acyl‐CoA:lysophosphatidylcholine acyltransferase in injured nerve, while choline kinase activity remained unchanged. This indicates that stimulation of phosphatidylcholine synthesis occurs via the cytidine nucleotide pathway, as well as by increased acylation of lysophosphatidylcholine. Although the cause of stimulated phosphatidylcholine synthesis remains unexplained, it is possible that trace amounts of lysophospholipids or other metabolites produced by injury‐enhanced phospholipase activity may be responsible.