bluuebunny/crossref_metadata_2025 huggingface dataset:
Modulatory Action of Taurine on the Release of GABA in Cerebellar Slices of the Guinea Pig
Authors: Namima M. , Okamoto K. , Sakai Y.
Published: December 2006

Abstract: For the purpose of demonstrating the action of taurine as a neuromodulator in addition to its suggested neurotransmitter function, the effects of taurine and muscimol on the depolarization‐induced Ca‐dependent release of [3H]γ‐aminobutyric acid (pH]GABA) and l‐[3H]glutamate in cerebellar slices from guinea pigs were investigated. The release of [3H]GABA was found to be greatly decreased by a GABA agonist, muscimol, and by taurine, but not by glycine. The release of l‐[3H]glutamate was little affected by taurine. The release of [3H]GABA was enhanced by bicuculline and strychnine, but not by picrotoxin, and the suppressive action of muscimol on the GABA release was antagonized by bicuculline, picrotoxin, and strychnine, suggesting the possible existence of presynaptic autoreceptors for GABA in the cerebellum. The suppressive action of taurine on the release of [3H]GABA, on the other hand, was blocked only by bicuculline. These results suggest that taurine reduced the release of [3H]GABA from cerebellar slices by acting on the GABA autoreceptors or, more likely, on other types of receptors that are sensitive to bicuculline. As a possible mechanism for this modulatory action of taurine, the blockade by this amino acid of the influx of Ca2+ into cerebellar tissues was tentatively suggested.
Changes of β‐Endorphin and Met‐Enkephalin Content in the Hypothalamus‐Pituitary Axis Induced by Aging
Authors: Missale C. , Govoni S. , Croce L. , Bosio A. , Spano P. F. , Trabucchi M.
Published: December 2006

Abstract: The amounts of β‐endorphin‐ and Met‐enkephalin‐immunoreactive material are higher in the pituitary of aged rats. However, the aging process decreases the content of βendophin‐, but does not affect that of Met‐enkephalin‐immunoreactive material, in hypothalamus. Thus, it seems that the regulatory mechanisms in the two areas are differentially affected by increasing age. On the other hand, the pituitary increase of these peptides is in line with the assumption that in the elderly the hormonal response to stress is impaired.
Changes in Rapid Transport of Phospholipids in the Rat Sciatic Nerve During Axonal Regeneration
Authors: Alberghina M. , Moschella F. , Viola M. , Brancati V. , Micali G. , Giuffrida A. M.
Published: December 2006

Abstract: Axonal transport of phospholipids in normal and regenerating sciatic nerve of the rat was studied. At various intervals after axotomy of the right sciatic nerve in the midthigh region and subsequent perineurial sutures of the transected fascicles, a mixture of 60 μCi [Me‐HC]choline and 15 μCi [2‐3H]glycerol in the region of the spinal motor neurons of the L5 and L6 segments was injected bilaterally. The amount of radioactive lipid (and in certain cases its distribution in various lipid classes) along the nerve was determined as a function of time. Three days after fascicular suture and 6 h after spinal cord injection of precursors, there was an accumulation of labeled phospholipids and sphingolipids in the transected sciatic nerve in the region immediately proximal to the site of suture. Nine days after, there was a marked increase in the accumulation of radioactivity in the distal segments of the injured nerve, which increased up to 14 days after cutting and disappeared as regeneration proceeded (21–45 days). In all segments of both normal and regenerating nerve fibers, as well as in L5 and L6 spinal cord segments, only phosphatidylcholine and sphingomyelin were labeled with [14C]choline. These results suggest that the regeneration process in a distal segment of a peripheral neuron, following cutting and fascicular repairing by surgical sutures, is sustained in the first 3 weeks by changes in the amount of phospholipids rapidly transported along the axon towards the site of nerve fiber outgrowth.
Effects of Light on Dopamine Metabolism in the Chick Retina
Authors: Parkinson David , Rando Robert R.
Published: December 2006

Abstract: The effect of prolonged exposure to light on the activity of dopaminergic neurons and dopamine (DA) metabolism of chick retinae was investigated. α‐Fluoromethyldopa, a potent and specific irreversible inactivator of aromatic amino acid decarboxylase, was used to assess DA turnover after inhibition of synthesis, and also to assess in vivo tyrosine hydroxylase activity by dihydroxyphenylalanine accumulation. After 48 h of light exposure, retinal DA in 12‐day‐old chicks was about 30% higher (p < 0.005) whereas dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) were elevated two to three times (p < 0.005) the level of controls kept in the dark for the same period. DA turnover was about twofold faster in the light (t1/2= 31 min) than in the dark (t1/2= 65 min). Tyrosine hydroxylase, assayed in vitro with saturating levels of cofactor and substrate, increased by about 50% after light exposure. The apparent tyrosine hydroxylase activity in vivo was approximately sixfold higher in the light than the dark. These results are interpreted and discussed in terms of the regulation of DA synthesis, and the use of DOPAC and HVA as indices of DA function in the retina.
P2 Protein in Oligodendrocytes and Myelin of the Rabbit Central Nervous System
Authors: Trapp Bruce D. , Itoyama Yasuto , MacIntosh Tracy D. , Quarles Richard H.
Published: December 2006

Abstract: P2 protein, a myelin‐specific protein, was detected immunocyto‐chemically and biochemically in rabbit central nervous system (CNS) myelin. P2 protein was synthesized by rabbit oligodendrocytes and was present in varying amounts throughout the rabbit CNS. Comparison of P2 and myelin basic protein (MBP) stained sections revealed that P2 antiserum did not stain all myelin sheaths within the rabbit CNS. The proportion of myelin sheaths stained by P2 antiserum and the amount of P2 detected biochemically were greater in more caudal regions of the rabbit CNS. The highest concentration of P2 protein was found in rabbit spinal cord myelin, where P2 antiserum stained the majority of myelin sheaths. P2 protein was barely detectable biochemically in myelin isolated from frontal cortex, and in sections of frontal cortex only occasional myelin sheaths reacted with P2 antiserum. These results suggest that the regional variations in the amount of P2 protein are due to regional differences in the number of myelin sheaths that contain P2 protein. P2 protein was detected immunocytochemically and biochemically in rabbit sciatic nerve myelin. Immunocytochemically, P2 antiserum only stained a portion of the myelin sheaths present. The myelin sheaths not reacting with P2 antiserum had small diameters and represented less than 10% of the total myelinated fibers.
Localization of Choline Acetyltransferase in Laminae of the Rat Olfactory Tubercle
Authors: Gordon Carin R. , Krieger Neil R.
Published: December 2006

Abstract: We report the distribution of choline acetyltransferase (ChAT) activity in the laminae of the rat olfactory tubercle. Within its posterior medial portion, the tubercle contains three parallel histological laminae that can be separated by cutting tangential sections from frozen tissue. ChAT was measured in homogenates of consecutive sections (16 μm) cut parallel to these laminae. The distribution of ChAT activity, as a function of tubercle depth, showed a broad peak centered at 500 μm from the ventral surface of the brain. Enzyme activity measured at this depth (85 pmol acetylcholine formed/μg protein/h) was 2 1/2 times greater than that measured in the outermost, plexiform, layer. Stereotaxic injections of kainic acid (1 μg in 1 μ1) made directly into the tubercle were used to eliminate intrinsic neurons. Three days after injection, histological examination revealed the almost total absence of neuronal cell bodies and the proliferation of glial cells. The greatest decreases in ChAT activity (50%) were seen at depths of 300–600 μm whereas no loss of activity occurred in the plexiform layer.
Effects of In Vivo Hypoxia on Acetylcholine Synthesis by Rat Brain Synaptosomes
Authors: Booth R. F. G. , Harvey S. A. K. , Clark J. B.
Published: December 2006

Abstract: Synaptosomes from normoxic and hypoxic rats were incubated aerobically in the presence and absence of veratridine. In the absence of veratridine, no significant difference was observed between the two types of preparation regarding either ATP/ADP ratio or 14CO2 or [14C]acetylcholine synthesis from D‐[U‐14C]glucose. However, in the presence of veratridine, significant reductions in the output of 14CO2 and [14C]acetylcholine by synaptosomes from hypoxic rats were apparent. It was concluded that irreversible metabolic lesions occur at the synapse as a result of hypoxia, which are apparent only when the metabolism of the preparation is accelerated to a level comparable with the maximal rate occurring in vivo. The presence of such lesions is further evidenced by the significant reductions in ATP/ADP ratio, 14CO2 output, and [14C]acetylcholine synthesis that occur in synaptosomes from hypoxic rats made anoxic in vitro and permitted to recover. Such decreases are not seen when synaptosomes from normoxic rats are similarly treated.
Regulation of Glycogenolysis in Transformed Astrocytes In Vitro
Authors: Cummins C. J. , Lust W. D. , Passonneau J. V.
Published: December 2006

Abstract: Cultured astrocytes, transformed by Herpesvirus, were used as a model system to study several aspects of the control of glycogenolysis. Adrenergic agonists such as norepinephrine and isoproterenol caused an immediate and dose‐dependent increase in the intracellular levels of cyclic AMP. Concomitant with the initial phase of cyclic AMP increase, conversion of phosphorylase b to a and glycogenolysis were observed. The elevation of cyclic AMP, phosphorylase conversion, and glycogenolysis were simultaneously blocked by β‐adrenergic blockers, but not by α‐adrenergic blocking agents. Repeated administration of norepinephrine caused an attenuated response in both cyclic AMP accumulation and glycogenolysis. Glycogen degradation is also partially regulated by glucose availability. In the presence of glucose, norepinephrine‐induced glycogenolysis is blocked, despite elevations in cyclic AMP. The direct role of glucose is postulated, since glucose analogs mimic the effects of glucose.
Neuraminidase in Calf Retinal Outer Segment Membranes
Authors: Dreyfus H. , Preti A. , Harth S. , Pellicone C. , Virmaux N.
Published: December 2006

Abstract: An enzyme catalyzing the hydrolysis of sialic acid (N‐acetylneuraminic acid: NeuNAc)‐containing glycoconjugates has been found in bovine retinal rod outer segment (ROS) membranes. The enzymatic activity is optimal at pH 4.0 and is stimulated by 0.15% Triton X‐100. Total activity was determined by the release of NeuNAc from endogenous and exogenous substrates (GDla). The ROS enzyme preferentially hydrolyses the ROS gangliosides, possibly because they are more accessible than the glycoproteins as substrates for the neuraminidase. Release of NeuNAc from gangliosides leads to important changes in the ganglioside patterns; whereas the amounts of GM1 increased throughout the incubation, the levels of polysialogangliosides GTlb and GD3 diminished owing to their rapid hydrolysis. The finding that gangliosides are hydrolysed more extensively than glycoproteins suggests that endogenous ROS gangliosides may be the principal source of metabolically available sialic acid in ROS. It was also observed that the activity of ROS neuraminidase is not affected by illumination of the membranes.
Immunohistochemical Localization of α‐Mannosidase During Postnatal Development of the Rat Cerebellum
Authors: Zanetta J. P. , Roussel G. , Dontenwill M. , Vincendon G.
Published: December 2006

Abstract: The localization of α‐d‐mannosidase in the rat cerebellum was studied by using indirect immunohistochemistry at both optical and electron microscopic levels. In the adult the enzyme is particularly concentrated in the dendrites and cell bodies of Purkinje cells, basket cells, and Golgi neurons in the cerebellar cortex and in the cytoplasm and dendrites of deep nuclei neurons. The cytoplasm of granule cells is poorly stained, whereas parallel fibers, white matter, Bergman fibers, and Golgi epitheloid cell perikarya show virtually no staining. Electron microscopy suggests that most of the staining is found in the cytosol, although some staining is found in the postsynaptic densities of the synapses between parallel fibers and Purkinje dendrites. The pattern of staining was followed throughout the postnatal development of the rat cerebellum. At birth an intense and diffuse staining is found in all cells except those of the external germinative layer. At the 6th postnatal day, Purkinje cell bodies and apical cones are strongly labeled. From the 13th day on, the pattern is very similar to that found in the adult. However, at the 18th postnatal day (when compared with the other structures), the staining of Purkinje cell dendrites seems to be higher than at all other ages. These data are correlated with biochemical studies and discussed in relation to the possible role of this enzyme during the postnatal development of the rat cerebellum.
MAO, COMT, and GABA‐T Activities in Primary Astroglial Cultures
Authors: Hansson Elisabeth , Sellström Åke
Published: December 2006

Abstract: Cultures from cerebral heimspheres of newborn rats contain the enzymes monoamine oxidase (MAO), catechol‐O‐methyltransferase (COMT), and α‐aminobutyric acid γ‐ketoglutarate transaminase (GABA‐T). The COMT activity was higher in the cultures than in adult rat cerebral hemispheres. The MAO activity was comparable in the cultures and in the rat cerebral hemispheres. The activities of both these enzymes increased with age in the cultures and in the rat brain hemispheres. In the culture the activities were further potentiated by removal of fetal calf serum and addition of 0.1 mM dibutyryl‐cyclic AMP (dB‐cAMP). GABA‐T activity was, however, lower in the cultures than in the adult rat brain hemispheres. The activity increased in brain during postnatal maturation. No changes in the enzyme activity were observed in the cultures, either during growth or after removal of fetal calf serum and addition of dB‐cAMP.
Clonidine Prevents Methylxanthine Stimulation of Norepinephrine Metabolism in Rat Brain
Authors: Galloway Matthew P. , Roth Robert H.
Published: December 2006

Abstract: Methylxanthines can produce behavior resembling opiate withdrawal in rats. Since previous studies have demonstrated the involvement of central noradrenergic systems during naloxone‐precipitated withdrawal, the effects of 3‐isobutyl‐l‐methylxanthine (IBMX) on norepinephrine metabolism in rat brain were studied. It was found that administration of IBMX elevated levels of the major norepinephrine metabolite 3‐methoxy‐4‐hydroxyphenyl‐glycol (MHPG) in areas innervated by the locus coeruleus. The increase in MHPG was noted 1 h after administration and was maximal (270% of control) after 3 h. Levels of another norepinephrine metabolite, 3,4‐di‐hydroxyphenylglycol, followed a similar pattern and time course. Coad‐ministration of naloxone with IBMX did not affect the IBMX‐induced elevation in MHPG. Administration of the a‐agonist clonidine, however, antagonized the effects of IBMX on MHPG levels. The effects of IBMX and clonidine were dose dependent; the lowest dose of IBMX needed to elevate MHPG was 30μmol/kg (i.p.), and clonidine (180 nmol/kg) reduced the effect of IBMX (100 μmol/kg) by 50%. The data, discussed in terms of a methylxanthine‐noradrenergic interaction, suggest that withdrawal behaviors in general may be subserved by hyperactive noradrenergic neurons.
Effects of CDPcholine and CDPethanolamine on the Alterations in Rat Brain Lipid Metabolism Induced by Global Ischemia
Authors: Dorman Robert V. , Dabrowiecki Zbigniew , Horrocks Lloyd A.
Published: December 2006

Abstract: The fast turnover pool of rat brain lipids was labeled by intracerebral injection of [3H]acetate. Cerebral ischemia for a duration of 5 min after decapitation caused a 2.2‐fold increase in radioactivity in the free fatty acids and loss of more than 20% of the radioactivity from choline and ethanolamine glycerophospholipids. An intracerebral injection of 0.6 μmol each of cytidine diphosphocholine (CDPcholine) and cytidine diphosphoethanolamine (CDPethanolamine) prevented the loss of radioactivity from the glycerophospholipids and decreased the amount of radioactivity in the free fatty acids by 59% as compared with control values and 82% as compared with ischemia values. By GLC assays of the mass of the free fatty acids, there was a threefold increase of free fatty acids in ischemic brains. Pretreatment of ischemic brains with CDPcholine and CDPethanolamine reduced the levels of unesterified fatty acids to 60% of the control values. Thus, a prior injection of cytidine nucleotides prevented the release of free fatty acids observed in ischemic brains.
Adenosine Metabolism in a Rat Hippocampal Slice Preparation: Incorporation into S‐Adenosylhomocysteine
Authors: Reddington Martin , Pusch Renate
Published: December 2006

Abstract: The incorporation of [14C]adenosine into various metabolites was studied in a hippocampal slice preparation in order to assess the extent of adenosine metabolism via synthesis of S‐adenosylhomocysteine, a potent inhibitor of transmethylation reactions. Highest incorporation of 14C occurred into nucleotides, with only a few percent being recovered in inosine + hypoxanthine, S‐adenosylhomocysteine, and the free adenosine pool. Labeling of S‐adenosylhomocysteine did not significantly increase with higher concentrations of added adenosine despite greater accumulation of free [14C]adenosine in the tissue. Addition of l‐homocysteine significantly increased the labelling of S‐adenosylhomocysteine. The results indicate that S‐adenosylhomocysteine synthesis is a minor pathway of adenosine metabolism in brain tissue under steady‐state conditions. Further, changes in adenosine concentration, without a concomitant change in l‐homocysteine availability, are unlikely to lead to a significant accumulation of S‐adenosylhomocysteine. S‐Adenosylhomocysteine is therefore not likely to play a significant role in mediating the biological effects of adenosine in the CNS via inhibition of transmethylations.
Regional Distribution of Catecholamines in Nucleus Accumbens of the Rabbit
Authors: DeFrance Jon F. , Sikes Robert W. , Gottesfeld Zehava
Published: December 2006

Abstract: The nucleus accumbens is an important telencephalic region, which is the target limbic and mesolimbic pathways. Because of an ongoing physiological study of the effects of dopamine, we wanted to determine regional differences of dopamine and norepinephrine concentrations in the nucleus. As determined by radioenzymatic assays, dopamine levels were not significantly different in the anterior‐posterior dimension, averaging approximately 187 ng dopamine/mg protein. Substantial amounts of norepinephrine were found throughout the nucleus, but the levels were significantly higher in the caudal portions of the nucleus, being approximately 4.5 times higher than in the anterior portions.
The Activity of 2′,3′‐Cyclic Nucleotide 3′‐Phosphohydrolase in the Corpus Callosum, Subcortical White Matter, and Spinal Cord in Infants Dying from Sudden Infant Death Syndrome
Authors: Carey Eric M. , Foster Peter C.
Published: December 2006

Abstract: The activity of 2′,3′‐cyclic nucleotide 3′‐phos‐phohydrolase (CNPase) has been determined in corpus callosum, subcortical white matter, and spinal cord of infants whose death was attributed to the sudden infant death syndrome (SIDS), and compared with enzyme activity in other cases in which the cause of death was not associated with respiratory distress. In nearly half the SIDS cases, CNPase activity and oligodendroglial cell numbers were reduced before the onset of myelination, but only in the corpus callosum. In other SIDS cases, enzyme activity and cell numbers were the same as in non‐SIDS cases. If the expression of CNPase activity reflects glioblast differentiation to oligodendrocytes with myelinating potential, then this transformation is abnormal in certain SIDS cases, as also evidenced in cases of prolonged neonatal respiratory insufficiency and gives rise to a subsequent deficit of myelin in the corpus callosum.
Stress‐Induced Enhancement of Suppression of [3H]GABA Release from Striatal Slices by Presynaptic Autoreceptor
Authors: Kuriyama Kinya , Kanmori Kouji , Taguchi Jun‐ichi , Yoneda Yukio
Published: December 2006

Abstract:The effect of cold and immobilization stress on presynaptic GABAergic autoreceptors was examined using the release of [3H]GABA (γ‐aminobutyric acid) from slices of rat striatum. It was found that in vitro addition of γ‐aminolevulinic acid, as well as GABA agonists such as muscimol and imidazoleacetic acid, exhibited a significant suppression of the striatal release of [3H]GABA evoked by the addition of high potassium, whereas γ‐aminovaleric acid had no significant effects on the evoked release. These suppressive actions were antagonized invariably by the GABA antagonists, bicucul‐line and picrotoxin, but not by the glycine antagonist, strychnine. Cholinergic agonists, such as pilocarpine and tetramethylammonium, also attenuated significantly the evoked release of [3H]GABA from striatal slices, while none of its antagonists, including atropine, hexame‐thonium and d‐tubocurarine, affected the release. On the other hand, in vitro addition of dopamine receptor agents such as dopamine, apomorphine, and haloperidol, or the inhibitory amino acids, glycine, γ‐alanine, and taurine failed to influence the evoked release of [3H]GABA from striatal slices. Application of a cold and immobilization stress for 3 h was found to induce a significant enhancement of the suppressive effects by muscimol and γ‐aminolevulinic acid on the evoked release of [3H]GABA, without affecting that by pilocarpine and tetramethylammonium. These results suggest that the release of GABA from striatal GABA neurons may be regulated by presynaptic autoreceptors for this neuroactive amino acid, and may play a significant functional role in the exhibition of various symptoms induced by stress.
Cholesterol Synthesis and Nerve Regeneration
Authors: Heacock Anne M. , Klinger Paul D. , Seguin Edward B. , Agranoff Bernard W.
Published: December 2006

Abstract: In this report, we examine the requirement of cholesterol biosynthesis and its axonal transport for goldfish optic nerve regeneration. Cholesterol, labeled by intraocular injection of [3H]mevalonolactone. exhibited a delayed appearance in the optic tectum. Squalene and other minor components were labeled but not transported. Following optic nerve crush, the amount of labeled cholesterol transport was elevated, while retinal labeling was not altered relative to control fish. A requirement for cholesterol biosynthesis is inferred from the inhibition of neurite outgrowth in retinal explants caused by the cholesterol synthesis inhibitor, 20, 25‐diazacholes‐terol. The inhibition of growth could be overcome by addition of mevalonolactone, but not cholesterol, to the medium. Intraperitoneal administration of 200 nmol of dia‐zacholesterol resulted in 92‐98% inhibition of retinal cholesterol synthesis and accumulation of labeled des‐mosterol and other lipids in fish retina and brain which persisted for 2 weeks. Diazacholesterol‐treated fish showed no reduction in the amount of lipid‐soluble radioactivity transported following intraocular injection of [3H]mevalonolactone, but there were alterations in the chromatographic pattern of the transported labeled lipids. In contrast to its effects on neurite outgrowth in vitro, diazacholesterol did not inhibit optic nerve regeneration in vivo, as measured both by arrival of labeled rapidly transported protein at the tectum and by time required for the return of visual function.
Purification and Characterization of a Membrane‐Bound Enkephalin‐Forming Carboxypeptidase, “Enkephalin Convertase”
Authors: Supattapone Surachai , Fricker Lloyd D. , Snyder Solomon H.
Published: December 2006

Abstract: Enkephalin convertase, the enkephalin‐synthe‐sizing carboxypeptidase B‐like enzyme, has been purified to apparent homogeneity from bovine pituitary and adrenal chromaffin granule membranes. The membrane‐bound enkephalin convertase can be solubilized in high yield with 0.5% Triton X‐100 in the presence of 1 M NaCl. Extensive purification is achieved by affinity chromatog‐raphy with p‐aminobenzoyl‐L‐arginine linked to Sepha‐rose 6B. Enzyme purified from both pituitary and adrenal chromaffin granule membranes shows a single band by sodium dodecyl sulfate polyacrylamide gel electrophoresis with an apparent molecular weight of 52,500, whereas enkephalin convertase purified from soluble extracts of these tissues has an apparent molecular weight of 50,000. The regional distribution of the membrane‐bound enzyme in the rat brain differs from that of the soluble enzyme. While the soluble enzyme shows 10‐fold variations, resembling somewhat the enkephalin peptides, membrane‐bound enkephalin convertase is more homogeneously distributed throughout the brain. In rat pituitary glands, membrane‐bound enzyme activity is similar in the anterior and posterior lobes, whereas the soluble enzyme is enriched in the anterior lobe. Membrane‐bound and soluble forms of enkephalin convertase isolated from either bovine pituitary glands or adrenal chromaffin granules show identical substrate and inhibitor specificities. As with the soluble enzyme, membrane‐bound enkephalin convertase hydrolyzes [Met]‐ and [Leu]enkephalin‐Arg6 and ‐Lys6 to enkephalin, with no further degradation of the pentapeptide.
Dolichol and Dolichyl Phosphate Levels in Brain Tissue from English Setters with Ceroid Lipofuscinosis
Authors: Kennedy Keller R. , Armstrong Donald , Crum Fred C. , Koppang Nils
Published: December 2006

Abstract: Human ceroid lipofuscinosis (CL) is an inherited disease marked by cerebromacular degeneration and early death. We have utilized the canine model to investigate the possible role of dolichol and dolichyl phosphate in the developmental pathology of CL. We found that while brain levels of dolichol increase with age in both affected and unaffected dogs, the amount in the diseased animal was similar to that in controls. Brain levels of dolichyl phosphate ranged from 20 to 35 μg/g in control dogs at all ages examined, but increased substantially during development in the affected dogs, a value of 113 ± 24 μg/g (mean ± SD) being obtained in the end‐stage animals. In addition to the results obtained in the canine model, dolichyl phosphate levels in human brain tissues from a 5‐year‐old with late infantile CL and from a 19‐year‐old with juvenile CL were found to be 153 and 382 μg/g, respectively, compared with a control that assayed 26 μg/g. The preliminary findings with human tissues provide further evidence for an association of elevated brain dolichyl phosphate levels with CL. Whether the increase is primary or secondary remains to be determined.
Glutamine Oxidation by Dissociated Cells and Homogenates of Rat Brain: Kinetics and Inhibitor Studies
Authors: Tyson Tildon J. , Roeder Lois M.
Published: December 2006

Abstract: The rates of [U‐14C]glutamine oxidation to 14CO2 were determined under a variety of experimental conditions using whole homogenates and dissociated cells from rat brain. The pattern of glutamine oxidation by homogenates differed from that by dissociated brain cells in several respects. The rates of glutamine oxidation by dissociated brain cells showed saturation kinetics with an apparent Km of 0.30 mM. Lineweaver‐Burk plots of glutamine oxidation by homogenates revealed two linear segments with two apparent Km values (0.58 mM and 3.0 mM). In the presence of aminooxyacetate, however, the Lineweaver‐Burk plots for homogenates were linear with a single Km of 0.47 mM. The oxidation of glutamine by homogenates was inhibited by both rotenone and anti‐mycin A (80‐85%), as were glutamate and glucose oxidation, suggesting that a significant amount of glutamine is oxidized via the tricarboxylic acid cycle. In the presence of aminooxyacetate, glutamine oxidation was inhibited <40%, whereas the oxidation of glutamate was inhibited 75%; in contrast, glucose oxidation was enhanced 50%. The rates of glutamine oxidation by homogenates were highest in the presence of high levels of potassium (50 mM) and low levels of sodium (2.5 mM). Varying ionic composition, however, had little or no effect on the rates of glutamine oxidation by dissociated brain cells. Measurements of glutamine oxidation by homogenates prepared from 2‐, 10‐, 15‐, 25‐, and 90‐day‐old rats revealed little or no age‐dependent difference. In contrast, the oxidation by dissociated brain cells from 2‐day‐old animals was significantly less than that obtained for animals 10 days or older (7.76 vs. 15.6 nmol/h/mg). These results suggest that a significant amount of glutamine can be oxidized via the tricarboxylic acid cycle and is a potential source of energy for the brain. The observed differences between the rates of glutamine oxidation by whole homogenates and dissociated brain cells suggest that the presence of an intact cell membrane may play a role in the regulation of glutamine oxidation. Alternatively, the results may reflect a difference in the concentration of effector(s) or intermediates, since the cellular constituents are more dilute in the homogenate preparation than in the intact cells. Another possibility is that these results may be indicative of a difference in the cellular and subcellular components present in the homogenates as compared with the dissociated cells.
High‐Affinity Neurotensin Binding Sites in Differentiated Neuroblastoma N1E115 Cells
Authors: Poustis Claudine , Mazella Jean , Kitabgi Patrick , Vincent Jean‐Pierre
Published: December 2006

Abstract: This paper describes the interaction of neurotensin with mouse neuroblastoma N1E115 cells. Neurotensin binding sites are undetectable in nondifferentiated neuroblastoma cells. They appear during cell differentiation in the presence of a low serum concentration and dimethyl sulfoxide, and reach a maximal level after 50–60 h of incubation under these conditions. The binding of monoiodo[Trp11]neurotensin to homogenates of differentiated N1E115 cells is specific, saturable, and reversible. The interaction is characterized by a dissociation constant of 150 pM and a maximal binding capacity of 9 fmol/mg of protein at 0°C, pH 7.5. These binding parameters, as well as the specificity toward a series of neurotensin analogues, are similar for neurotensin receptors in N1E115 cells and for the high‐affinity binding sites that had been previously characterized in rat brain synaptic membranes by means of the same radiolabeled ligand. The presence of high‐affinity binding sites for neurotensin in the neuroblastoma N1E115 provides a useful model to study the cellular responses that are generated by the association of neurotensin to its receptor in electrically excitable cells.
Cell‐Cycle Dependence of a Ganglioside Glycosyltransferase Activity and Its Inhibition by Enkephalin in a Neurotumor Cell Line
Authors: Scheideler Mark A. , Lockney Michael W. , Dawson Glyn
Published: December 2006

Abstract: Rat glioma mouse neuroblastoma hybrid neurotumor cells (NG108‐15), synchronized by amino acid deprivation, showed a cell‐cycle‐dependent peak of activity of a ganglioside N‐acetylgalactosaminyl transferase 14‐24 h following release from the cell cycle block (S/G2 phase). Maximal expression of two typical lysosomal hydrolases, N‐acetyl‐β‐hexosaminidase and β‐galactosidase, occurred between 18 and 21 h following release (S phase), declining to G1 phase levels during the peak of N‐acetylgalactosamine (GalNAc) transferase activity. In addition, glycosyltransferase activity in G2 phase cells showed an increase in apparent Vmax (suggesting the presence of more enzyme/mg of cell protein) and apparent binding affinity for uridine diphosphate N‐acetylgalactosamine (UDP‐GalNAc) (32 versus 14 M) when compared to transferase activity in the G1 phase. However, the opioid peptide enkephalin [D‐Ala2, o‐Leu5], which inhibits ganglioside GalNAc transferase activity in unsynchronized NG108‐15 cultures, was much more inhibitory in whole cells 8 h after release from the cell cycle block (G1 phase) than in cells 20 h after release (G, phase), with 50% inhibition occurring at 2 ± 10‐9M and 2 ± 10‐7M, respectively. These results suggest that the GalNAc transferase activity is regulated in more than one way during the cell cycle, since both Vmax and Km changes are observed, and that the cyclic AMP‐dependent mechanism by which opiates reduce transferase activity is receptor mediated and cell cycle dependent.
Extraction Techniques for Gastrins and Cholecystokinins in the Rat Central Nervous System
Authors: Marley Philip D. , Rehfeld Jens F.
Published: December 2006

Abstract: Samples of rat striatum and synthetic sul‐phated cholecystokinin octapeptide were extracted by different procedures and the solubilised cholecystokinin‐like immunoreactivity analysed by gel filtration and ion‐exchange chromatography. Ice‐cold 90% methanol extraction gave the greatest recovery of tissue immunoreactivity without any major modification of the extracted components. The 33‐amino acid form of cholecystokinin was poorly recovered by this extractant. Boiling water or a combined boiling water/acetic acid extraction gave efficient recovery of tissue immunoreactivity but chemically modified a substantial part of the octapeptide‐like component. Boiling in acetic acid alone also produced this modification but in addition resulted in poor recovery of the octapeptide‐like component. The combined water/ acetic acid extraction gave reasonable to good recovery of all added Cholecystokinins and gastrins, including the 33‐amino acid form of cholecystokinin. Ice‐cold 0.1 M HCl was less efficient than 90% methanol at solubilising tissue immunoreactivity and resulted in a substantial modification of the octapeptide component distinct from that produced by boiling extractions, possibly desulpha‐tion. The results show that more than one extraction procedure is needed to study all the cholecystokinin components in brain tissue and demonstrates the necessity of using at least two chromatographic systems for such studies.
Distribution and Chromatographic Characterisation of Gastrin and Cholecystokinin in the Rat Central Nervous System
Authors: Marley Philip D. , Rehfeld Jens F. , Emson Piers C.
Published: December 2006

Abstract: Two tissue extraction techniques and two radioimmunoassays were used to study the distribution of gastrin and cholecystokinin in rat brain. Small amounts of gastrin were found in extracts of neurohypophysis, but in neither ice‐cold 90% methanol nor in boiling water‐acetic acid extracts of the other 33 brain areas studied. Cholecystokinin was found in equivalent amounts in both types of extract of 31 areas. The distribution was similar to that in previous studies. The components of cholecystokinin immunoreactivity were characterised in 10 rat CNS tissues using four tissue extraction methods in conjunction with gel filtration and ion‐exchange chromatography. The results demonstrated that gastrins were present only in the neurohypophysis and that in all other rat CNS tissues the main molecular component was indistinguishable from the sulphated octapeptide of cholecystokinin. Minor immunoreactive components were observed in all types of extract of all tissues with the properties of the desulphated octapeptide and the C‐terminal tetrapeptide amide, suggesting they are genuine tissue components, not extraction artefacts. Large molecular forms of cholecystokinin were not detected in any tissue. The results emphasise the necessity of using two or more extraction methods and two or more chromatography systems in such a study.
The Calmodulin Antagonists, Trifluoperazine and R24571, Depolarize the Mitochondria Within Guinea Pig Cerebral Cortical Synaptosomes
Authors: Snelling Rosalind , Nicholls David
Published: December 2006

Abstract: The effects of trifluoperazine and l‐[bis(p‐chlorophenyl)methyl] ‐ 3 ‐ [2,4 ‐ dichloro ‐ 3 ‐ (2,4 ‐ dichloroben‐zyloxy)phenethyl]imidazolium chloride (R24571) upon synaptosomal calcium transport, plasma membrane potential, in situ mitrochondrial membrane potential, and ATP levels are investigated in order to assess the suitability of these calmodulin antagonists for investigating calmodulin‐dependent processes in the nerve terminal. Both agents appear to act selectively at the mitochondrial membrane, causing extensive depolarization at concentrations in excess of 10 μM (trifluoperazine) or 0.5 μM (R24571). The extent of Ca uptake into the synaptosomes is decreased, consistent with the loss of the mitochondrial compartment. There is no inhibition of the efflux of Ca from the synaptosomes. Depolarization‐dependent Ca uptake is not prevented by R24571. Synaptosomal ATP levels decrease to an extent consistent with the collapse of the mitochondrial potential. It is concluded that the uncoupling effect of these agents on the in situ mitochondria prevents their being used to investigate the role of calmodulin in intact synaptosomes.
Alterations of Amino Acid Transmitter Systems in Spinal Cords of Chronic Paraplegic Dogs
Authors: McBride W. J. , Hall P. V. , Chernet E. , Patrick J. T. , Shapiro S.
Published: December 2006

Abstract: The high‐affinity uptake of [3H]serotonin, [3H]glutamate, and [3H]‐γ‐aminobutyric acid (3H]GABA) and the Na+‐independent binding of [3H]glutamate and [3H]GABA were studied using spinal cord preparations obtained from normal mongrel dogs and from dogs made paraplegic by midthoracic spinal cord crush. Lumbosa‐cral regions of the spinal cord were removed either before (1 week) or after (3 to 8 weeks) onset of spasticity. A myelin‐free synaptosomal fraction was obtained by cen‐trifugation and used for studying high‐affinity uptake and for preparing synaptic plasma membranes for Na+‐inde‐pendent binding experiments. For the paraplegic groups, the uptake of 30 nM [3H]serotonin was 66 and 18% of control values after 1 and 3 weeks, respectively. Eadie‐Hofstee analysis of [3H]serotonin uptake showed a 90% reduction in Vmax for the paraplegic group relative to control values, thereby indicating the expected loss of descending serotonergic pathways. The high‐affinity uptakes of 1 μM [3H]glutamate and [3H]GABA were the same in both the control and nonspastic paraplegic groups after 1 week. However, after 3 weeks, the uptakes of [3H]glutamate and [3H]GABA were 60‐70% higher for the spastic group than for the control animals. For both amino acids, Eadie‐Hofstee plots revealed no difference in Km and higher Vmax for the spastic group relative to control values. After 1 and 3 weeks, the Na+ ‐independent binding of 5 nM [3H]glutamate was 40‐85% higher and the binding of 10 nM [3H]GABA was 40‐60% lower for the paraplegic groups relative to the values for the control animals. Scatchard analysis revealed significant changes in Bmax values for both amino acids. Overall, the data indicate an increase in segmental amino acid excitatory influence which occurred when signs of spasticity were evident.
Forskolin Stimulates Adenylate Cyclase Activity, Cyclic AMP Accumulation, and Adrenocorticotropin Secretion from Mouse Anterior Pituitary Tumor Cells
Authors: Heisler Seymour , Reisine Terry
Published: December 2006

Abstract: The effects of forskolin, an adenylate cyclase activator, were investigated on adrenocorticotropin (ACTH) secretion from AtT‐20/D16‐16 mouse pituitary tumor cells. Forskolin increased adenylate cyclase activity in these cells in the absence of added guanyl nu‐cleotide, an effect blocked by somatostatin. Cyclic AMP synthesis and ACTH secretion increased in a concentration‐dependent manner, not only in the clonal cells, but in primary cultures of rat anterior pituitary as well. Somatostatin inhibited cyclic AMP synthesis and ACTH secretion in response to forskolin. When forskolin was coapplied with corticotropin releasing factor, cyclic AMP synthesis was potentiated and ACTH secretion additive. The calcium channel blocker, nifedipine, inhibited forskolin, and 8‐bromocyclic AMP stimulated ACTH secretion. These data suggest that ACTH secretion may be regulated at the molecular level by changes in cyclic AMP formation, which in turn regulate a calcium gating mechanism.
Characterization of Calcium‐Activated and Magnesium‐Activated ATPases of Brain Nerve Endings
Authors: Lin Shih‐chia C. , Way E. Leong
Published: December 2006

Abstract: The properties of Ca2 + ‐activated and Mg2 + ‐activated ATPases of nerve endings from mouse brain were investigated. Ca2 + and Mg2 + each can activate ATP hydrolysis in synaptosomes and its subfractions. Both Ca2 + ‐ATPase and Mg2 + ‐ATPase exhibit high and low affinity for their respective cations. At millimolar concentrations of Ca2 + or Mg2 +, several nucleoside triphosphates could serve as substrate for the two enzymes and their specific activities were about three to four times higher in synaptic vesicles than in synaptosomal plasma membranes (SPM). Both in SPM and in synaptic vesicles the relative activity in the presence of Ca2 + was in the order of CTP > UTP > GTP = ATP, but with Mg2 + the activity was higher with ATP than with the other three triphosphates. Mg2 +‐ATPase was more active than Ca2 + ‐ATPase in SPM, but in synaptic vesicles the two enzymes exhibited similar activity. Kinetic studies revealed that Mg2 + ‐ATPase was inhibited by excess ATP and not by excess Mg2 +. The simultaneous presence of Na++ K+ stimulated Mg2 + ‐ATPase and inhibited Ca2 + ‐ATPase activity in intact synaptosomes and SPM. The stimulation of Mg2 + ‐ATPase by Na2++ K+ was further increased by increasing Mg2 + concentration and was inhibited by Ca2 + and by ouabain. When Ca2 + and Mg2 + are present together in SPM or synaptic vesicles, the total Pi liberated by the two cations may either increase or decrease, depending on their relative concentrations. Kinetic analyses indicate that Ca2 + and Mg2 + bind independently to the enzyme alone or together at different sites. The results suggest that Ca2 + ‐ATPase and Mg2 + ‐ATPase in SPM or synaptic vesicles may be separate and distinct systems.
Selective Effects of Proteases and Phospholipase A2 on Monoamine Oxidases A and B of Human Brain and Liver
Authors: White Helen L. , Stine Deborah K.
Published: December 2006

Abstract: Human brain and liver mitochondria contain membrane‐bound monoamine oxidase of both A and B types. Monamine oxidase‐A (MAO‐A), either membrane‐ bound or in detergent‐solubilized extracts from these tissues, was selectively inhibited during incubations with trypsin, chymotrypsin, thermolysin, or papain. MAO‐A in solubilized, but not in membrane‐bound, preparations was also very sensitive to the action of phospholipase A2, while MAO‐B was unaffected. Membrane‐bound MAO‐ A of rat brain mitochondria was more sensitive to phospholipases and less sensitive to proteases than was human brain enzyme, indicating that these agents may reveal species differences in MAO properties. Human brain and liver MAO‐A, either solubilized or bound in mitochondrial membranes, apparently contains basic and aromatic peptide moieties that are available to proteases. Hydrolysis of these peptide bonds leads to rapid denaturation unless substrate molecules stabilize the activesite. Phospholipase A2 may disrupt the phospholipid microenvironment of MAO‐A, the integrity of which is essential for MAO‐A activity, but not for MAO‐B. No in‐ terconversion of the two activities was observed. After phospholipase A2 treatment, remaining MAO‐A activity was recovered in low‐molecular‐weight regions of a gel filtration gradient, suggesting that MAO‐A subunits were released. Although these experiments argue against the proposal that phospholipids may regulate the ratio of A/ B activities of a single enzyme molecule, it is conceivable that endogenous phospholipases or proteases in mitochondrial membranes may influence MAO‐A activity independently of MAO‐B activity.
Cleavage of Myelin Basic Protein by Neutral Protease Activity of Human White Matter and Myelin
Authors: Berlet Hans H. , Ilzenhöfer Heike , Schulz Günther
Published: December 2006

Abstract: Polypeptides arising from neutral in vitro proteolysis of myelin basic protein (MBP) of human brain were evaluated by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis. At pH 7 a marked breakdown of MBP resulted in the formation of 8–12 polypeptides ranging from 6 to 17 kd in molecular weight. As neutral proteolytic activity was not eliminated by either gel filtration or cation‐exchange chromatography acid‐soluble protease(s) involved probably have a size and electric charge similar to that of MBP. The enzymatic nature of neutral proteolysis was ascertained by heat inactivation and inhibition by α2‐macroglobulin. Incomplete inhibition of proteolysis and the failure of small peptides (< 6 kd) to show up on electrophoresis seem to suggest that MBP was degraded by exopeptic proteases as well. Acid extracts of purified myelin yielded polypeptides similar to those of MBP of delipidated white matter. The results are consistent with a sequential limited proteolysis of MBP by neutral proteases probably associated with myelin and possibly related to the in situ catabolism of MBP in man.
Some Characteristics of Glutamic Acid Decarboxylase of Chick Ampullary Cristae
Authors: Meza Graciela
Published: December 2006

Abstract: In a previous study, it was demonstrated that enzyme‐mediated γ‐aminobutyric acid (GABA) synthesis occurs in the vestibule of the chick inner ear. As deeper knowledge of the properties of its synthesizing enzyme might contribute to the understanding of the role of GABA in inner ear function, some characteristics of glutamate decarboxylase (GAD) were studied in chick isolated ampullary cristae under conditions in which 14CO2 release from [1‐14C]glutamate and [14C]GABA formation from [U‐14C]glutamate for estimating GAD activity were equal. It was found that Km for glutamate is 5 mM and that the enzyme pH optimum is 7.3. These values fall within the range described for the corresponding enzyme in nervous tissue of other species. Pyridoxal phosphate (PLP) activates the enzyme and aminooxyacetic acid inhibits it, the same as these agents activate or inhibit GAD from several nervous tissue sources. 2‐Mercaptoethanol shows some protection from inactivation of the PLP‐de‐pendent enzyme and Triton X‐100 exerts some inhibition of vestibular GAD activity, as previously shown in other nervous tissue preparations. Although its cellular localization is at present uncertain, these results indicate that GAD of chick vestibular tissue possesses properties resembling those of the brain enzyme and might be controlled in a manner similar to that of GAD in brain, thus possibly participating in the regulation of inner ear function.