bluuebunny/crossref_metadata_2025 huggingface dataset:
Endogenous Protein Phosphorylation in Rat Brain Mitochondria: Occurrence of a Novel ATP‐Dependent Form of the Autophosphorylated Enzyme Succinyl‐CoA Synthetase
Authors: Steiner Anton W. , Smith Roberts A.
Published: December 2006

Abstract: When rat brain mitochondria are incubated with [γ‐32P]ATP, there is a rapid (10 s) phosphorylation of proteins designated E, and F of M.W. 42,000 and 32,000, respectively. Although [γ‐32P]ATP was the preferred substrate for protein F, a small amount of labeling did occur with [γ‐32P]GTP. Phosphorylation of E1 was absolutely ATP‐dependent. On the other hand, a 32,000 M.W. protein from rat liver mitoplasts (mitochondria devoid of an outer membrane) was highly phosphorylated when [γ‐32P]GTP was used but not at all phosphorylated within short time periods with [γ‐32P]ATP. Both the ATP‐labeled brain phosphoprotein F and GTP‐labeled liver protein migrated to identical positions on high‐resolution two‐dimensional polyacrylamide gels, and both contained acid‐labile phosphoryl groups. Furthermore, both phosphoproteins were identified as the autophosphorylated subunit of succinyl‐CoA synthetase (SCS, EC 6.2.1.4) by using antibody directed against purified GTP‐dependent porcine SCS. However, immunotitration experiments with anti‐porcine SCS revealed that ATP‐ and GTP‐labeled protein F in brain differed in their interactions with antibody, suggesting that in rat brain mitochondria two different forms of the enzyme exist that are immunologically distinct and differ in substrate specificity. When mitochondrial preparations enriched in particular brain cell or subcellular types were examined, an unequal distribution of E1 and the two forms of protein F were observed. A brain subfraction containing neuronal cell body and glial mitochondria (CM) was found to contain E1 and approximately equal amounts of the ATP‐ and GTP‐dependent forms of protein F. Light synaptic mitochondria(SM1) contained ATP‐dependent protein F almost exclusively and were depleted in E1. Dense synaptic mitochondria (SM2) are rich in the ATP form of SCS but also contain low amounts of the GTP enzyme.
Increased Levels of Neuron‐Specific Enolase in PC 12 Pheochromocytoma Cells as a Result of Nerve Growth Factor Treatment
Authors: Vinores S. A. , Marangos P. J. , Parma A. M. , Guroff G.
Published: December 2006

Abstract: Treatment of PC 12 pheochromocytoma cells with nerve growth factor (NGF) resulted in increased levels of neuron‐specific enolase (NSE). Neither insulin, growth hormone, cytochrome c, nor sodium butyrate increased NSE levels. Epidermal growth factor (EGF) did increase NSE levels, although not to the same extent as NGF. As little as 1 ng/ml NGF induced the maximal increase in NSE. As PC 12 cells increased in density, the NSE levels increased even in untreated cells.
Ability of Human Leukocytic Pyrogen to Stimulate Brain Prostaglandin Synthesis In Vitro
Authors: Dinarello Charles A. , Bernheim Harry A.
Published: December 2006

Abstract: Fever is thought to be mediated by leukocytic pyrogen (LP), a polypeptide synthesized by phagocytic leukocytes and which is responsible for the upwards resetting of the hypothalamic thermostat. In an attempt to study the effects of LP directly on brain tissue, purified human LP was incubated with rabbit brain slices in vitro. Because of the well‐documented role of prostaglandin (PG) synthesis in both the production of fever and antipyresis, PGE levels were measured on the supernates of brain slices incubated 30 min with LP. Levels of PGE increased 3‐ to 4‐fold in rabbit anterior and posterior hypothalami. In addition, PGE levels were similarly increased in temporal cortex slices when exposed to LP. In another set of experiments, PGE levels increased 4‐ to 5‐fold when brain tissue was incubated with a highly purified preparation of bacterial endotoxin (ET). The ability of ET to increase brain PGE levels was not affected by moderate heating (56°C, 30 min), whereas this temperature destroyed the PGE‐inducing properties of LP. The antipyretic ibuprofen markedly reduced the amount of PGE measured in the brain slice supernates after stimulation with LP, suggesting that LP brings about synthesis of PGE and not the release of preformed PG. The results demonstrate that LP is a potent inducer of PGE synthesis in rabbit brain and that receptors for LP are not restricted to the thermoregulatory center, but rather may be distributed throughout the brain.
Benzodiazepine Antagonist Ro 15–1788: Binding Characteristics and Interaction with Drug‐Induced Changes in Dopamine Turnover and Cerebellar cGMP Levels
Authors: Möhler H. , Burkard W. P. , Keller H. H. , Richards J. G. , Haefely W.
Published: December 2006

Abstract: The recently discovered benzodiazepine antagonist Ro 15‐1788 was characterized in binding studies, and its potency and selectivity were determined in vivo by interaction with drug‐induced changes in dopamine turnover and cerebellar cGMP level. Ro 15‐1788 reduced [3H]flunitrazepam binding in the brain in vivo with a potency similar to that of diazepam and effectively inhibited [3H]diazepam binding in vitro (IC50= 2.3 ± 0.6 nmol/liter). [3H]Ro 15‐1788 bound to tissue fractions of rat cerebral cortex with an apparent dissociation constant (KD) of 1.0 ± 0.1 nmol/liter. The in vitro potency of various benzodiazepines in displacing [3H]Ro 15‐1788 from its binding site was of the same rank order as found previously in [3H]diazepam binding. Autoradiograms of [3H]Ro 15‐1788 binding in sections of rat cerebellum showed the same distribution of radioactivity as with [3H]flunitrazepam. The attenuating effect of diazepam on the chlorpromazine‐ or stress‐induced elevation of homovanillic acid in rat brain was antagonized by Ro 15‐1788. Among a series of compounds which either decreased or increased the rat cerebellar cGMP level, only the effect of benzodiazepine receptor ligands (diazepam, zopiclone, CL 218 872) was antagonized by Ro 15‐1788. Thus, Ro 15‐1788 is a selective benzodiazepine antagonist acting at the level of the benzodiazepine receptor in the central nervous system. Peripheral benzodiazepine binding sites in kidney and schistosomes were not affected by Ro 15‐1788.
Dysmyelination of Shiverer Mutant Mice In Vivo and In Vitro
Authors: Nagaike Kazuhiro , Mikoshiba Katsuhiko , Tsukada Yasuzo
Published: December 2006

Abstract: Dysmyelination in the CNS of shiverer mutant mice was studied in vivo and in vitro. By immunohistochemical reaction with glial fibrillary acidic protein antibody, hypertrophy of the fibrous astrocytes was observed in the white matter of shiverer cerebella. The cerebella of shiverer mice in primary culture from the day of birth showed very poor myelination under optical microscopy. Axons of Purkinje cells are thought to be the main myelinated axons in the primary culture of the cerebellum. Purkinje cells from shiverer appeared normal with regard to Bodian silver impregnation, hematoxylin and eosin staining, and P400 protein characterization of Purkinje cells. Addition of the conditioned culture medium of shiverer to the control culture did not interfere with myelination. We concluded that the dysmyelination in the CNS of shiverer could be caused by an intrinsic defect of the oligodendrocyte rather than by hypertrophy of the astrocytes or by diffusible factors.
Immunological Studies of Human Monoamine Oxidases
Authors: Brown G. K. , Powell J. F. , Craig I. W.
Published: December 2006

Abstract: Antisera have been raised against monoamine oxidase preparations from human placenta and platelets. These antisera have been employed to characterize membrane‐bound enzyme from a variety of human sources including liver, heart, and brain. The comparisons were based on a displacement radioimmunoassay system with soluble placenta! monoamine oxidase, previously labelled specifically with [3H]pargyline, as antigen. All forms of enzyme investigated demonstrated immunological cross reaction; however, the placental enzyme appeared to possess determinants not exhibited by the enzyme from the platelets or other tissues examined.
A Single Radioimmunological Assay for Serotonin, N‐Acetylserotonin, 5‐Methoxytryptamine, and Melatonin
Authors: Geffard M. R. , Puizillout J. J. , Delaage M. A.
Published: December 2006

Abstract: A radioimmunoassay of melatonin using a new iodinated derivative has been developed. Simple chemical treatments have then been designed to convert serotonin, N‐acetylserotonin, and 5‐methoxytryptamine to melatonin. Thus these four molecules, belonging to the same metabolic pathway, were separately assayed in the same radioimmunological system at the same sensitivity level (0.01 pmol). Some biological results on the circadian variations of melatonin and serotonin in blood, pineal, and miscellaneous brain structures are presented.
Modification of Brain Mitochondrial Enzymes by the Oxygen Analogue of Ronnel at Various Stages of Development and Aging
Authors: Sitkiewicz Dariusz , Skonieczna Maria , Rychla Tatiana , Gaździk Danuta , Podymniak Stefania , Bicz Włodzimierz
Published: December 2006

Abstract: This paper describes the effect of the organophosphorus compound, the oxygen analogue of ronnel (OAR), on the activity of some membrane‐bound enzyme systems in the brain mitochondria of developing, young‐adult, and old rats. Age‐related changes were noted in the cholesterol‐to‐protein ratio, whereas the phospholipid content in mitochondria showed little change during development as well as aging. The results obtained suggest that development of brain succinate dehydrogenase may consist in a decrease of Km and increase of Vmax values. In aged rats an altered, perhaps inhibited form of the enzyme is produced. The oxygen analogue of ronnel caused a mixed‐type inhibition of the succinate dehydrogenase derived from brains of 4‐day‐old, 16‐day‐old and 2‐month‐old animals. In the case of enzyme from the brain of 18‐month‐old rats, a typical competitive‐type inhibition was observed. Mechanisms responsible for inhibition of the succinate:cytochrome c reductase from brains of developing animals are similar to those for succinate dehydrogenase. In aged rats (18 months old), however, a noncompetitive mechanism of inhibition of succinate:cytochrome c reductase was revealed. The experiments reported here provide evidence that lipid‐soluble molecules of OAR may interact with membrane phospholipids and lead to modification of membrane architecture and also of enzyme kinetic behaviour. It may be also concluded, that the sensitivity of the enzyme systems studied to inhibition by OAR is an age‐dependent phenomenon. Modification of membrane by development or aging alters the kinetics as well as the sensitivity of enzymes to inhibitors.
Conjugated Dopamine in Superfusates of Slices of Rat Striatum
Authors: Tyce Gertrude M. , Rorie Duane K.
Published: December 2006

Abstract: An acid‐hydrolyzable conjugate of 3,4‐dihydroxyphenylethylamine (dopamine, DA) was detected in superfusates from slices from rat striatum. The concentrations of endogenous free and conjugated DA, and of the acid metabolites (3,4‐dihydroxyphenylacetic acid [DOPAC] and homovanillic acid [HVA]) in superfusates were measured using HPLC with electrochemical detection. Conjugated DA in superfusates represented 10–20% of the free DA under basal conditions and during release evoked by p‐tyramine (5 × 10−6M to 5 × 10−4M); much smaller amounts of conjugated DA overflowed into superfusate when DA was released by equimolar concentrations of β‐phenylethyl‐amine. Surprisingly, inhibition of monoamine oxidase by the inhibitors N‐methyl‐N‐propargyl‐3‐(2,4‐dichlorophenoxy)propylamine hydrochlo‐ride (clorgyline) or N‐methyl‐N‐2‐propynylbenylamine (pargyline) had little effect on the amounts of conjugated DA present in superfusate. Under basal conditions, the amounts of conjugated DA in superfusate were always less than the amounts of DOPAC but quite similar to the amounts of HVA. However, during release of DA evoked by p‐tyramine the concentrations of conjugated DA in superfusate showed much more pronounced increases than those of the acidic metabolites.
[3H]Dopamine Labeling of D3 Dopaminergic Sites in Human, Rat, and Calf Brain
Authors: List Stephen J. , Seeman Philip
Published: December 2006

Abstract: The binding of [3H]dopamine to brain regions of calf, rat, and human was investigated. The calf caudate contained the highest density of [3H]dopamine binding sites, with a Bmax value of 185 fmol/mg protein, whereas rat and human striatum contained one‐third this number of sites. The KD values for [3H]dopamine in all tissues were 2–3 nM. Dopaminergic catecholamines (dopamine, apomorphine, 6,7‐dihydroxy‐2‐aminotetralin, and N‐propylnorapomorphine) inhibited the binding of [3H]dopamine in all three species, at low concentrations, with IC50 values of 1.5 to 6 nM. Neuroleptics, in contrast, inhibited the binding at high concentrations (with IC50 values of 200 to 40,000 nM). The [3H]dopamine binding sites were saturable, heat‐labile, and detectable only in dopamine‐rich brain regions; these sites differed from D2 dopamine sites (labeled by [3H]butyrophenone neuroleptics), and from Dl dopamine sites (labeled by [3H]thioxanthene neuroleptics) associated with the dopamine‐stimulated adenylate cyclase. We have, therefore, called these high‐affinity [3H]dopamine binding sites D3 sites. [3H]Apomorphine and [3H]ADTN also appeared to label D3 sites. These ligands however, were less selective than [3H]dopamine, and labeled sites other than D3 as well. Assay conditions were important in determining the parameters of [3H]dopamine binding. The optimum conditions for selective labeling of the D3 dopaminergic sites, using [3H]dopamine, required the presence of EDTA and ascorbate.
Normalization of Cerebellar RNA Synthesis and mRNA Levels After Treatment of Graft Versus Host Disease
Authors: Griffin W. Sue T. , Snider B. Joy , Morrison Marcelle R.
Published: December 2006

Abstract: We have previously shown that neonatal rats with graft versus host disease (GVHD) (1) synthesize significantly less cerebellar RNA, (2) have RNA that is less translationally active, and (3) have changes in the relative abundance of certain mRNAs, including the induction of one coding for protein r that is present neither in control cerebellum nor in other brain regions at any age. Here we report on the ability of the cerebellum to recover from GVHD‐induced changes in the synthesis of total RNA and in the relative levels of specific mRNAs. In order to halt the disease, 11‐day‐old diseased Fischer animals were injected with hyperimmune alloantiserum daily for 3 days. Cytoplasmic RNAs were isolated from the cerebella of 14‐day‐old serum‐treated animals, their diseased littermates that were not treated with serum, and litter‐mate controls. Comparison, by two‐dimensional gel analysis, of the in vitro synthesized mRNA translation products showed that most GVHD‐induced alterations in the levels of specific mRNAs were not present in serum‐treated animals. In particular, protein r was not synthesized by cerebellar RNAs isolated from serum‐treated animals. These results show that the adverse effects of this disease are reversible at the molecular level.
Enkephalins and Opiate Antagonists Control Calmodulin Distribution in Neuroblastoma‐Glioma Cells
Authors: Baram Dana , Simantov Rabi
Published: December 2006

Abstract: The calcium binding protein calmodulin and the opiate receptor binding sites are unevenly distributed in various subcellular fractions of neuroblastoma‐glioma NG108‐15 cells. The crude mitochondrial‐membrane fraction of these cells contains two membrane fractions that are separable by sucrose gradient centrifugation. These two differ in the content of both calmodulin and opiate receptors. Leucine enkephalin and d‐Ala2‐methionine enkephalinamide decrease the amount of membrane‐bound calmodulin in the NG108‐15 cells in a time‐ and dose‐dependent manner, whereas the opiate antagonists naloxone and levallorphan have an opposite effect. Naloxone blocks the effect of leucine enkephalin and dextrallorphan has no significant effect. The opiate alkaloids entorphine and phenazocine induce changes similar to that of the enkephalins whereas morphine is inactive even at high concentrations. The alteration in the amount of membrane‐bound calmodulin after a short incubation (15 min) with the enkephalins or with naloxone is reflected as an opposite change in the amount of calmodulin in the cell cytosol. Naloxone and levallorphan also increase the number of opiate receptors in NG108‐15 cells but dextrallorphan has no such effect. Modulation of the intracellular distribution of calmodulin by opioid peptides and alkaloids may control the activity of various membrane‐bound and cytosolic systems that are calmodulin‐ and/or calcium‐dependent.
Serum Albumin Washing Specifically Enhances Arachidonate Incorporation into Synaptosomal Phosphatidylinositols
Authors: Strosznajder Joanna , Foudin Laurie , Tang Wilson , Sun Grace Y.
Published: December 2006

Abstract: Arachidonate incorporation into synaptosomal phospholipids was shown to be affected by factors including the procedure for preparation of the membrane fractions and preincubation of synaptosomes prior to assay of incorporation activity. Preincubation of synaptosomes gave rise to a decrease in incorporation of arachidonate into both phosphatidylcholine (PC) and phosphatidylinositol (PI). However, the inhibition toward incorporation into PIs, but not PCs, was fully reversed when the membranes were washed with bovine serum albumin. A twofold increase in arachidonate incorporation into PIs was also observed when freshly prepared synaptosomes were washed with serum albumin immediately before assay of incorporation activity. The inhibitory action is thought to be due to an increase in polyunsaturated fatty acids and/or their oxidation products which may then elicit a special effect on the acyltransferase responsible for transferring arachidonate into phosphatidylinositols. The differences in fatty acid uptake and response to serum albumin also suggest the presence of different acyltransferases for acyl transfer to PIs and PCs.
Carbonic Anhydrase, 5′‐Nucleotidase, and 2′,3′‐Cyclic Nucleotide‐3′‐Phosphodiesterase Activities in Oligodendrocytes, Astrocytes, and Neurons Isolated from the Brains of Developing Rats
Authors: Snyder D. Stephen , Zimmerman Thomas R. , Farooq Muhammad , Norton William T. , Cammer Wendy
Published: December 2006

Abstract: The activities of three myelin‐associated enzymes, carbonic anhydrase, 5′‐nucleotidase, and 2′,3′‐cyclic nucleotide‐3′‐phosphodiesterase (CNP), were measured in oligodendrocytes, neurons, and astrocytes isolated from the brains of rats 10, 20, 60, and 120 days old. The carbonic anhydrase specific activity in oligodendrocytes was three‐ to fivefold higher than that in brain homogenates at each age, and, at all the ages, low activities of this enzyme were measured in neurons and astrocytes. The oligodendrocytes and astrocytes from the brains of rats at all ages had higher activities of the membrane‐bound enzyme 5′‐nucleotidase than was observed in neurons. In oligodendrocytes from 10‐ and 20‐day‐old rats, the 5′‐nucleotidase activity was two‐ to threefold the activity in the homogenates (i.e., relative specific activity = 2.0–3.0), and the relative specific activity of this enzyme in the oligodendrocytes declined to less than 1.0 at the later ages, concomitant with the accumulation of 5′‐nucleotidase in myelin. The CNP activity was always higher in oligodendrocytes than in neurons, but not appreciably different from that in astrocytes from 20 days of age onward. The relative specific activity of CNP was highest in the oligodendrocytes from 10‐day‐old rats but was lower, at all ages, than we had observed in bovine oligodendrocytes. These enzyme activities in oligodendroglia are quite different in amount and developmental pattern from those reported previously for myelin.
Adenosine Triphosphatase Activity at the External Surface of Chicken Brain Synaptosomes
Authors: Nagy Ágnes , Shuster Terry A. , Rosenberg Murray D.
Published: December 2006

Abstract: An ATP‐hydrolysing activity on the external surface of intact synaptosomes from chicken forebrain has been investigated. The observed ATPase activity was not due to leakage of the intracellular ATPase activities, to artefacts resulting from breakage of the nerve endings during the incubation and isolation periods, or to possible contamination by other subcellular particles. Disruption of the synaptosomes resulted in an approximately 2.5‐fold increase of the basal, Mg2+‐dependent ATPase activity, suggesting that the plasma membrane was acting as a permeability barrier to the substrate. ATP hydrolysis was maximal (0.8 μumol Pi/min/mg protein) at pH 8.2 in a medium containing either Mg2+ or Ca2+ ions. Ouabain (0.2 mM) and oligomycin (2 μg/mg protein) had no appreciable effect on this ATPase activity. Kinetic studies of the enzyme revealed an apparent Km value for ATP of approximately 4 ± 10−5M. These data are consistent with the view that the observed ATP hydrolysis was being catalysed by an ectoenzyme, i.e., an enzyme in the plasma membrane of the nerve endings with its active site facing the external medium. The rapid hydrolysis of the released ATP is a suspected function for this ecto‐ATPase.
3Development‐Dependent Regulation of N‐Acetyl‐β‐d‐Hexosaminidase of Cerebellum and Cerebrum of Normal and Staggerer Mutant Mice
Authors: Wille W. , Heinlein U. A. O. , Spier‐Michl I. , Thielsch H. , Trenkner E.
Published: December 2006

Abstract: Distinctive activities of various glycosidases were expressed in the cerebellum and cerebral cortex of mice during their development. In particular, N‐acetyl‐β‐D‐hexosaminidase (EC 3.2.1.30) appeared to be developmentally regulated. A transient peak of enzyme activity at postnatal day 7 was characteristic for the cerebellum, whereas the activity in the cerebral cortex gradually increased through the 1st postnatal month and was maintained at a high level of activity throughout adulthood. The regulation of Af‐acetylhexosaminidase activity in the developing cerebellum of the staggerer mouse deviated clearly from enzyme activities in the wild‐type, whereas the activity pattern in the staggerer cerebral cortex remained unaffected. In experiments mixing wild‐type and staggerer cerebellum homogenates, the specific activity was additive. Thus, involvement of inhibitors or activating molecules can be excluded. This developmentally controlled regulation or disregulation in staggerer appears to be enzyme specific, since β‐glucosidase, α‐glucosidase, and β‐galactosidase did not exhibit such a pattern in either normal or staggerer mice. In the mutation weaver that, like staggerer, loses the majority of its cerebellar granule cells, N‐acetyl‐β ‐hexosaminidase activity of the cerebellum was not elevated, indicating a specific defect in staggerer rather than a general effect on lysosomal enzymes due to cell death.
Reversibility of Propranolol‐Induced Changes in the Biosynthesis of Monoacylglycerol, Diacylglycerol, Triacylglycerol, and Phospholipids in the Retina
Authors: Ilincheta de Boschero Monica G. , Bazan Nicolas G.
Published: December 2006

Abstract: The biosynthesis and metabolism of phospholipids and neutral glycerides were studied in the bovine retina. Radioactive glycerol was used as a precursor. Phentolamine and d‐ and dl‐propranolol were found to produce similar effects on lipid metabolism in the retina. Marked stimulation of phosphatidylinositol (PhI) synthesis and maximal inhibition of phosphatidylcholine (PhC), diacylglycerol (DG), and triacylglycerol (TG) formation were observed within 5 min after exposure to 0.5 mM dl‐propranolol. Pulse‐chase experiments showed a high turnover rate in DG and a reversibility of the propranolol‐induced changes produced during the synthesis of PhC, TG, DG, monoacylglycerol (MG), and phosphatidylserine. All reversals of the drug‐induced biosynthetic profiles approached control values 60 min after incubation in drug‐free medium. However, complete reversal was not achieved in any of the cases under these conditions. Propranolol appeared to inhibit both the formation of DG from phosphatidic acid and the further metabolism of DG, probably to MG. Phosphatidylethanolamine biosynthesis showed some recovery from this inhibition. Synthesis of Phi was greatly stimulated by preincubation with propranolol and was further enhanced by reincubation in the presence of propranolol. However, this effect was not reversed by reincubation without the drug. The active de novo biosynthesis of retinal phospholipids and glycerides is a very dynamic pathway that may be redirected by amphiphilic drugs. In addition, the partial reversal of modifications induced in the flux of [2‐3H]glycerol through the lipids can occur during short‐term reincubations of retinas in drug‐free medium.
Inhibition by Diazepam and γ‐Aminobutyric Acid of Depolarization‐Induced Release of [14C]Cysteine Sulfinate and [3H]Glutamate in Rat Hippocampal Slices
Authors: Baba Akemichi , Okumura Shigetoshi , Mizuo Hiroyuki , Iwata Heitaroh
Published: December 2006

Abstract: Effects of diazepam and γ‐aminobutyric acid‐related compounds on the release of [14C]cysteine sulfinate and [3H]glutamate from preloaded hippocampal slices of rat brain were examined by a superfusion method. Diazepam markedly inhibited the release of cysteine sulfinate and glutamate evoked either by high K+ or veratridine without affecting that of other neurotransmitter candidates, e.g., γ‐aminobutyric acid, acetylcholine, noradrenaline, and dopamine; IC50 values for the release of cysteine sulfinate and glutamate were about 20 and 7 μM, respectively. γ‐Aminobutyric acid (1 to 10 μM) and muscimol (100 μM) significantly reduced high K+‐stimulated release of glutamate. Bicuculline, which had no effect on the release at a concentration of 50 μM by itself, antagonized the inhibitory effects of diazepam and γ‐aminobutyric acid on glutamate release. Similar results were obtained with the release of cysteine sulfinate except that a high concentration (100 μM) of γ‐aminobutyric acid was required for the inhibition. These results indicate the modulation by γ‐aminobutyric acid innervation of the release of excitatory amino acids in rat hippocampal formation, and also suggest that some of the pharmacological effects of diazepam may be a consequence of inhibition of excitatory amino acid transmission.
Vectorial Discharge and Localization of Nascent Polypeptides in Rough Endoplasmic Reticulum of Developing Neuronal Perikarya of Rat Brain Cortex
Authors: Khawaja Jahangir A. , Lindholm Dan B.
Published: December 2006

Abstract: Rough endoplasmic reticulum (RER) prepared from bulk‐isolated neuronal perikarya of rat brain cortex of different postnatal ages was found to be active in vectorial discharge of nascent proteins through the membrane; this activity increased with the increasing age of animals and reached maximal values in adults. RER isolated from whole cortical tissue (containing all cell types) exhibited vectorial release only up to 18 days of age; the preparation from adult animals was essentially devoid of secretory activity. Controlled proteolysis of various preparations suggested that in neuronal RER of 8‐day‐old rats the proportion of nascent proteins operationally retained in the intravesicular space was about twice that retained by cortical preparations. For the purpose of comparison, these parameters were studied also in liver RER.
Characterization of the Binding of [3H]Ro 5‐4864, a Convulsant Benzodiazepine, to Guinea Pig Brain
Authors: Weissman Ben Avi , Bolger Gordon T. , Isaac Lawrence , Paul Steven M. , Skolnick Phil
Published: December 2006

Abstract: The density of high affinity binding sites for [3H]4′‐chlorodiazepam ([3H]Ro 5‐4864) in guinea pig cerebral cortex is significantly higher (3.8‐fold) than the density reported in the rat, and is nearly equal to the density of binding sites for other [3H]benzodiazepines (e.g., diazepam, flunitrazepam). The density of these [3H]Ro 5‐4864 binding sites was generally higher in guinea pig brain than in rat brain, with the exception of olfactory bulb. Both the subcellular distribution and pharmacologic profile of these sites in guinea pig brain appears qualitatively similar to observations previously reported in the rat. The high density of binding sites for [3H]Ro 5‐4864, coupled with the potency of this compound as a convulsant in the guinea pig, suggest this species will be a valuable model for elucidating putative pharmacologic and physiologic functions of these sites in brain.
Accumulation of Thyrotropin Releasing Hormone by Rat Hypothalamic Slices
Authors: Charli Jean‐Louis , Ponce Georgina , McKelvy Jeffrey F. , Joseph‐Bravo Patricia
Published: December 2006

Abstract: It has been postulated that thyrotropin releasing hormone (TRH) may play an active role in synaptic transmission. If such is the case, an inactivation mechanism must exist, in analogy to other neuroactive substances. In these studies we have considered the possibility that TRH may be taken up by rat hypothalamic slices. We observed that in the presence of bacitracin TRH was stable in the medium up to 90 min. We detected intact [3H]Pro‐TRH associated with the slices as evidenced by TLC and paper electrophoresis; the association was time‐dependent up to 60 min, and the maximum tissue‐to‐medium ratio was 1.3 at this time. At 5 min incubation, 30‐50% of the TRH was not extracellular, and the plot of TRH‐associated tissue versus the total amount of tissue was linear up to two hypothalami per flask. The association was saturable (Km 1.07 μM) and temperature‐dependent, and the saturable part of the accumulation was inhibited by ouabain, dinitrophenol, and the absence of glucose. These results suggest that an uptake mechanism for TRH exists in the hypothalamus; its physiological relevance remains to be elucidated.
Specificity and Sodium Dependence of the Active Nucleoside Transport System in Choroid Plexus
Authors: Spector Reynold , Huntoon Sheryl
Published: December 2006

Abstract: The transport of [3H]deoxyuridine by the active nucleoside transport system into the isolated rabbit choroid plexus was measured in vitro under various conditions. Choroid plexuses were incubated in artificial CSF containing 1 μM [3H]deoxyuridine and 1 μM nitroben‐zylthioinosine for 5 min under 95% O2‐5% CO2 at 37°C and the accumulation of [3H]deoxyuridine measured. Ni‐trobenzylthioinosine was added to the artificial CSF at a concentration (1 μM) that did not inhibit the active nucleoside transport system but did inhibit the separate, saturable nucleoside efflux system. The active transport of deoxyuridine into the choroid plexus depended on Na + in the medium, as ouabain, substitution of Li+ and cho‐line for Na+, and poly‐L‐lysine all inhibited deoxyuridine transport. Thiocyanate in place of chloride and penetrating sulfhydryl reagents also inhibited the active transport of deoxyuridine into choroid plexus. The active transport of deoxyuridine into choroid plexus, which is inhibited by naturally occurring ribo‐ and deoxyribonu‐cleosides (IC50= 7‐21 μM), was not inhibited (IC50 > 150 μM) by nucleosides with certain alterations on the 2′, 3′, or 5’positions in D‐ribose or 2‐deoxy‐D‐ribose (e.g., adenine arabinoside, 3′‐deoxyadenosine, xylosyl‐adenosine); or the pyrimidine or purine rings (e.g., 6‐azauridine, xanthosine, 7‐methylinosine, or 8‐bromo‐adenosine). Other analogues were effective (IC50= 8‐26 μM; e.g., 5‐substituted pyrimidine nucleosides, 7‐deaza‐adenosine, 6‐mercaptoguanosine) or less effective (IC50= 46‐145 μM; e.g., 5‐azacytidine, 3‐deazauridine) inhibitors of deoxyuridine transport into the isolated choroid plexus.
Arginine Metabolism in Mouse Brain Synaptosomes
Authors: Johnson Jeffery L. , Roberts Eugene
Published: December 2006

Abstract: In a study employing mouse brain synaptosomes and synaptosomal sonicates, the complete metabolic machinery was found to be present for transport of arginine into synaptosomes, its conversion to ornithine, and the formation from the latter of glutamic acid, γ‐aminobutyric acid, and proline. The results show that a delicate balance probably exists between the flows of metabolites. This balance, which probably determines the steady‐state levels of these substances in nerve terminals, can be altered by concentrations of the metabolites themselves through feedback inhibition as well as by levels of cofactors.
Activation of Choline Acetyltransferase by Vasoactive Intestinal Peptide
Authors: Luine Victoria N. , Rostene William , Rhodes Joseph , McEwen Bruce S.
Published: December 2006

Abstract: Addition of vasoactive intestinal peptide (VIP) to brain homogenates increased the activity of choline acetyltransferase (ChAT) but not that of acetylcholinesterase or glucose‐6‐phosphate dehydrogenase. Activity of ChAT was increased in the anterior hypothalamus and in the dorsal and ventral hippocampus, but not in the parietal cortex or posterior hypothalamus. Increased activity occurred rapidly after VIP addition to homogenates and was maximal at 10 ‐7M concentration. Kinetic analysis indicates that the Vmax of the enzyme is increased and the Km for choline, but not acetyl‐coenzyme A, is decreased in the presence of VIP. Results support a possible VIP‐cholinergic interaction in the CNS.
Ornithine‐σ‐Aminotransferase Exhibits Different Kinetic Properties in Astrocytes, Cerebral Cortex Interneurons, and Cerebellar Granule Cells in Primary Culture
Authors: Drejer Jørgen , Schousboe Arne
Published: December 2006

Abstract: To evaluate a possible role of ornithine‐γ‐ami‐notransferase (EC 2.6.1.13; Orn‐T) as a rate‐limiting enzyme for the synthesis of transmitter glutamate and γ‐aminobutyric acid (GABA), respectively, its activity and kinetic properties were analyzed in cultured astrocytes as well as in neuronal cultures consisting mainly of glutamatergic neurons (cerebellar granule cells) or GABAergic neurons (cerebral cortex interneurons). For comparison the activity and kinetics of Orn‐T were also assayed in mouse brain homogenates. The highest activity of Orn‐T was found in astrocytes and in cerebral cortical neurons (5.3 ± 0.5 and 5.3 ± 0.4 nmol mg‐1 min‐1, respectively) whereas the activities of Orn‐T in cerebellar granule cell cultures and in mouse brain were found to be about half of these values (3.1 ± 0.3 and 2.8 ± 0.1 nmol min‐1 mg‐1, respectively). From a kinetic study of Orn‐T in the different preparations only a relatively low affinity for the enzyme with respect to ornithine was found in cerebellar granule cells, astrocytes, and whole brain [apparent Km values (at 0.5 mM γ‐ketoglutarate): 4.7 ± 0.9, 4.3 ± 2.2, and 6.8 ± 2.2 mM, respectively] whereas the corresponding Km value for Orn‐T in cerebral cortex interneurons was found to be significantly lower (apparent Km: 0.8 ± 0.3 mM). The enzyme was not found to be inhibited by GABA (range 0.1‐10 mM) in any of the preparations.
Proteins of the Brain Extracellular Fluid: Evidence for Release of S‐100 Protein
Authors: Shashoua Victor E. , Hesse Gary W. , Moore Blake W.
Published: December 2006

Abstract: Extracellular protein fractions were obtained (1) by mild, isotonic irrigation of freshly perfused brain tissue; (2) by collection of proteins released into super‐fusing medium by physiologically viable slices of rat hippocampus; and (3) by sampling the CSF of anesthetized rats. Analysis of the S‐100 protein content of these fractions gave values of 2.8, 4.2, and 1.8 μg S‐100/mg protein, respectively. These values were three‐ to sixfold higher than the S‐100 content of the soluble cytoplasmic protein fractions from the same tissue. This several‐fold higher S‐100 content of the extracellular protein fractions relative to the intracellular cytoplasmic protein fractions indicates that S‐100 is selectively released into the extracellular spaces of the brain. We suggest that the biological function of this CNS protein may involve intercellular transfer.
Binding of ATP to Brain Glutamate Decarboxylase as Studied by Affinity Chromatography
Authors: Wu Susan J. , Martin David L.
Published: December 2006

Abstract: The interactions of two forms of porcine brain glutamate decarboxylase (β‐GAD and γ‐GAD) with the effector ATP were studied by affinity chromatography. A third form, γk‐GAD, was only slightly retarded by the affinity matrix and was eluted in the buffer wash. The interaction of GAD with the ATP affinity matrix was qualitatively similar to its interaction with free ATP as reported in previous kinetic studies. The rank order of adenine nucleotides as eluting agents and affinity ligands was ATP > ADP > AMP. GAD was also eluted by its cofactor, pyridoxal 5′‐phosphate, and this was enhanced by 1 mM Pi In contrast, a high concentration (140 mM) of Pi by itself was required to elute the enzyme. GAD remained active while bound to the affinity column and was eluted in the holoenzyme form by ATP, indicating that the affinity ligand did not bind in the active site and did not displace catalytically active cofactor from the enzyme.
Nucleoside and Oxypurine Homeostasis in Adult Rabbit Cerebrospinal Fluid and Plasma
Authors: Eells Janis Thane , Spector Reynold , Huntoon Sheryl
Published: December 2006

Abstract: In adult New Zealand white rabbits, the effects of food deprivation and of massive elevations of plasma uridine or thymidine concentrations on CSF and plasma nucleoside and oxypurine concentrations were studied. Nucleoside and oxypurine levels were determined by high performance liquid chromatography using unequivocal methods of compound identification. After 48 and 96 h of food deprivation, the concentrations of uridine, cytidine, inosine, thymidine, deoxycytidine, deoxyuridine, hypoxanthine, xanthine, and uric acid in CSF and plasma were not different than in controls, except at 96 h, when the plasma uridine concentration was 35% lower (p < 0.05). After elevation of the plasma and CSF thymidine concentrations to ∼200 and 100 μM, respectively, with intravenous thymidine for 5 h, there was a large increase in CSF and plasma thymine to ∼100 μM and a smaller increase in plasma and CSF deoxyuridine concentrations. After elevation of the plasma and CSF uridine concentrations to 0.6 and 0.2 mM, respectively, there was a large increase in CSF and plasma uracil and a smaller increase in plasma and CSF deoxyuridine concentrations. Elevated plasma concentration of thymidine and uridine significantly decreased the CSF to plasma ratios of deoxyuridine and thymidine; however, only elevated plasma uridine concentrations decreased the CSF to plasma ratio of uridine. These results document the powerful homeostatic mechanisms that regulate the concentrations of the principal nucleosides and oxypurine bases in CSF.
Muscarinic Receptor During Postnatal Development of Rat Cerebellum: An Index of Cholinergic Synapse Formation?
Authors: Mallol J. , Sarraga M. C. , Bartolome M. , Ghandour M. S. , Gombos G.
Published: December 2006

Abstract: Quantitative and qualitative modifications of the specific binding sites for [3H]quinuclidinyl benzylate (QNB), a muscarinic antagonist, were studied during rat cerebellar postnatal development. Specific binding sites for QNB (QNB‐sbs), regardless of whether they correspond to muscarinic acetylcholine receptors, are present with the highest density in the archicerebellar cortex, but the total amount per region is about the same in the archi‐, paleo‐, and neocerebellar cortex regions. Large amounts of QNB‐sbs are also present in a cerebellar fraction including central white matter and deep cerebellar nuclei. QNB‐sbs are low but present at birth and then accumulate during ontogenic development according to a curve which duplicates, with a delay of a few days, the curve of DNA accumulation. Dissection studies indicated that this curve does not depend on the preferential localization of QNB‐sbs in a specific cerebellar region nor on the particular development of this region. The similarity of the QNB‐sbs and the DNA developmental curves might indicate that the QNB‐sbs are present on granule cells; however, a comparative analysis of the data in the literature suggests that a great many QNB‐sbs are located on the Purkinje cell dendrites in the molecular layer, where all or some of them might correspond to the ex‐trajunctional muscarinic acetylcholine receptor detected there by electrophysiology. It would appear that only a small percentage of cerebellar QNB‐sbs corresponds to the cholinergic synapses present in cerebellar cortex; hence, the question of muscarinic receptors in the cerebellum should be re‐examined.
Conjugates of Dopamine and Serotonin in Ventriculocisternal Perfusates of the Cat
Authors: Hammond Donna L. , Yaksh Tony L. , Tyce Gertrude M.
Published: December 2006

Abstract: The in vivo formation of acid‐hydrolyzable conjugates of dopamine and of serotonin (presumably dopamine‐‐O‐‐sulfate and serotonin‐O‐sulfate) in ventriculo‐ cisternal perfusions of the cat is described. Small amounts of these amine conjugates were detected under quiescent conditions and during evoked release of the parent amines. The amounts of conjugated dopamine in perfusate were increased during and immediately after the period in which release of dopamine was evoked, but were not affected by inhibition of monoamine oxidase. In contrast, the efflux of serotonin conjugate during the evoked release of serotonin was not increased unless monoamine oxidase was inhibited. The data suggest that conjugation of amines in the CNS may be of functional importance in their disposition either under conditions of augmented release or during inhibition of oxidative deamination.
Involvement of Endo‐Oligopeptidases A and B in the Degradation of Neurotensin by Rabbit Brain
Authors: Camargo A. C. M. , Almeida M. L. C. , Emson P. C.
Published: December 2006

Abstract: Incubation of neurotensin with rabbit brain cytosol fractions resulted in a rapid loss of carboxy‐terminal neurotensin immunoreactivity, whereas the amino‐terminal portion of neurotensin was relatively stable to degradation. Neurotensin degradation by rabbit brain cystosol fractions was activated by dithiothreitol and inhibited by thiol blocking reagents, Zn2 +, and by monospecific antibodies against endo‐oligopeptidases A and B, thus suggesting the possible involvement of these enzymes in neurotensin degradation by rabbit brain. An association of a proportion of endo‐oligopeptidase B activity (proline endo‐peptidase) with the membrane fraction of nervous tissue was suggested by the presence of proline endopeptidase activity on the membranes and by immunoprecipitation of the solubilized activity using antiendo‐oligopeptidase B antiserum.
2′,3′‐Cyclic Nucleotide 3′‐Phosphodiesterase and 5′‐Nucleotidase in the Central Nervous System of a Myelin‐Deficient Rat Mutant
Authors: Hof Liselotte , Csiza Charles K. , Dentinger Mark P. , Barren Kevin D.
Published: December 2006

Abstract: 2′,3′‐Cyclic nucleotide 3′‐phosphodiesterase activity was examined in brains and spinal cords of normal and myelin‐deficient Wistar rats. While the activity in normal brains increased from 0.2 μ‐mol/min/mg protein (units) at 6–10 days to 3.5 units at 25 days of postnatal age, the activity in the myelin‐deficient rat remained at 0.2–0.3 units over the same period. In spinal cord, the normal activities were 5.7 and 10.9 units at 12 and 20 days, respectively, whereas they declined in the myelin‐deficient rat from 1.06 to 0.79 units for the same age points. 5′‐Nucleotidase activities in brain and spinal cord were normal in the myelin deficient rat at both ages.
Genetic Association Between Ca2+‐ATPase Activity and Audiogenic Seizures in Mice
Authors: Palayoor Sanjeewani T. , Seyfried Thomas N.
Published: December 2006

Abstract: Ca2+ ‐ATPase activity was studied in fresh brain stem homogenates of the audiogenic seizure (AGS)‐resistant C57BL/6 and AGS‐susceptible DBA/2 inbred strains and in 21 B6 χ D2 recombinant inbred strains. A highly significant negative correlation was found between Ca2+ ‐ATPase activity and AGS susceptibility among these strains. In general, strains with low Ca2+‐ATPase activities were more AGS‐susceptible than strains with high activities. Further, Ca2+ ‐ATPase activity appears to be influenced by a major gene associated with the Ah locus. This gene is designated Caa for Ca2+ ‐ATPase activity and is different from las, which is closely linked to the Ah locus. las influences AGS spread by a yet unknown biochemical mechanism, whereas Caa may influence AGS susceptibility by regulating Ca2+‐ATPase activity in brain tissue.
Characterization of 125I‐Lysergic Acid Diethylamide Binding to Serotonin Receptors in Rat Frontal Cortex
Authors: Kadan Michael J. , Krohn Ann Marie , Evans Mark J. , Waltz Robin L. , Hartig Paul R.
Published: December 2006

Abstract: 125I‐Lysergic acid diethylamide (125I‐LSD) is the first 125I‐labeled ligand for serotonin receptor studies. Its binding to rat frontal cortex membranes is saturable, reversible, and stereospecific. Specific binding is linearly dependent on tissue concentration and represents 70–80% of the total binding. Scatchard plots of the binding data are linear with a KD of 1.5 nM, a Bmax of 12.4 fmol/mg wet weight tissue, and a Hill slope of 1.02. The binding kinetics are highly temperature‐dependent. At 37°C the bimolecular association rate constant is 1.28 × 108 min−1M−1 and the dissociation rate constant is 0.087 min−1 (t1/2= 8.0 min). At 0°C < 4% dissociation occurs over 40 min and the association rate is similarly depressed. Inhibition of 125I‐LSD binding by a variety of serotonergic, dopaminergic, and adrenergic ligands reveals a 5‐hydroxytryptamine2 (5‐HT2) serotonergic profile for this binding site. Regional distribution studies of 125I‐LSD binding in rat brain show that areas with the highest levels of binding include the cortex and striatum. Iodinated radioligands can be synthesized with specific activities exceeding 2,000 Ci/mmol, which makes them approximately 75‐fold more sensitive than tritiated radioligands. This high specific activity, coupled with the selectivity of 125I‐LSD for 5‐HT2 sites, makes this ligand a sensitive new probe for 5‐HT2 serotonin receptors.
Incorporation of [14C]Glucose into Lipids of Bovine Oligodendroglia
Authors: Gibson A. , Brammer M. J.
Published: December 2006

Abstract: Oligodendrocytes were isolated from the white matter of ox brains. Light microscopy revealed that the cells were ≥90% phase‐bright with a mean diameter of 7.6 μm. Transmission electron microscopy was employed to identify the classic morphology associated with mature oligodendrocytes. Homogenates of the isolated cells showed negligible activity of neuronal and astrocytic cell markers. Using a suspension culture method cells were incubated with [14C]glucose. This simple precursor labelled the five complex lipids choline glycerophospholipid, ethanolamine glycerophospholipid, inositol glycerophospholipid + serine glycerophospholipid, and the two cerebroside species. The incorporation of label was shown to be dependent on glucose concentration, protein concentration, and the length of incubation. In addition the glucose uptake blocker phloretin (1 mM) reduced the degree of labelling by up to 97%, and the metabolic poisons KCN (1 mM) and iodoacetate (0.5 mM) had varying deleterious effects on the amounts of labelling of the five lipids measured.