bluuebunny/crossref_metadata_2025 huggingface dataset:
Myelin Deficits Produced by Early Postnatal Exposure to Inorganic Lead or Triethyltin Are Persistent
Authors: Toews Arrel D. , Blaker William D. , Thomas David J. , Gaynor Jeffrey J. , Krigman Martin R. , Mushak Paul , Morell Pierre
Published: October 2006

Abstract: Long‐Evans rat pups were exposed to either inorganic lead (400 mg Pb as lead acetate/kg body weight/day) or triethyltin sulfate (1.0 mg/kg body weight/day), by gastric intubation, from 2 days through 29 days of age. The rats were then weaned and placed on standard lab chow ad libitum. At 30 days of age, leadtreated rats exhibited statistically significant decreases in body and brain weights (22% and 17%, respectively), and the concentration of forebrain myelin was significantly reduced, by 21% relative to the 4.9 mg myelin protein/g brain in control rats. Although these animals recovered from the body weight deficits after several months, the deficits in brain weight and myelin concentration were still present at 120 days of age. This suggests that the lead‐induced myelin deficits were permanent. Lead levels in brain, which were maximal at 30 days of age when the treatment was terminated, decreased more slowly than in other organs and were still 30% of maximal levels at 120 days of age. Triethyltin‐treated animals also had significantly decreased body and brain weights (20% and 11%, respectively) at 30 days of age, and an even more severe reduction in forebrain myelin concentration (33%). These animals also regained a normal body weight by 120 days of age, but again the deficits in brain weight and myelin concentration persisted. Tin levels in brain and other organs had decreased to control levels by 60 days of age. Animals malnourished by maternal deprivation to match the body weights of the treated animals had myelin deficits that were less severe than those in the treated animals at 30 days of age (approximately 11% less than controls); however, these myelin deficits also persisted throughout the subsequent 90‐day recovery period examined. The apparent lack of recovery from CNS myelin deficits produced by neonatal exposure to different heavy metals or to malnutrition reemphasizes the vulnerability of the developing nervous system to a wide range of metabolic insults.
Cortical GABA Turnover During Bicuculline Seizures in Rats
Authors: Chapman Astrid G. , Evans Mary C.
Published: October 2006

Abstract: The rate of cortical γ‐aminobutyric acid (GABA) turnover was estimated by determining the rate of GABA accumulation following inhibition of GABA transaminase by γ‐vinyl‐GABA (1.5 g/kg, i.v.) in paralysed, ventilated rats. During 1 h of bucuculline‐induced seizures (1.2 mg/kg, i.v.) the rate of accumulation of cortical GABA level is approximately threefold greater than in the control group receiving γ‐vinyl‐GABA alone, suggesting that the GABA shunt activity increases in parallel with the increase in overall cortical metabolic rate observed during bicuculline seizures. Pretreatment with γ‐vinyl‐GABA did not affect the bicuculline‐induced changes in other major cortical amino acids.
Effect of Denervation and Reinnervation on Oxidation of [6‐14C]Gucose by Rat Skeletal Muscle Homogenates
Authors: DuBois Debra C. , Max Stephen R.
Published: October 2006

Abstract: We studied the effects of denervation and reinnervation of the rat extensor digitorum longus muscle (EDL) on the oxidation of [6‐14C]glucose to 14CO2. The rate of 14CO2 production decreased dramatically following denervation, and the decrease became significant 20 days after nerve section. Prior to day 20, changes apparently reflected the decline of muscle mass. Decreased 14CO2 production was due to reduced capacity of the enzymatic system (apparent Vmax); there was no change in apparent affinity for glucose (apparent Km). Mixing experiments revealed that the loss of oxidative capacity following denervation is not caused by production of soluble inhibitors by degenerating muscle. Oxidative metabolism, as measured by 14CO2 evolution, recovered during reinnervation. Surprisingly, the specific activity in reinnervated muscles displayed an “overshoot” of approximately 50%, which returned to control by day 60, possibly reflecting increased energy demand by the growing muscle. The time‐course of the denervation‐mediated change indicates that altered oxidative capacity is secondary to events that initiate denervation changes in muscle. Nevertheless, diminished oxidative capacity may be of considerable metabolic significance in denervated muscle.
A Highly Sensitive Enzyme Immunoassay for Mouse β Nerve Growth Factor
Authors: Furukawa Shoei , Kamo Isao , Furukawa Yoshiko , Akazawa Saeko , Satoyoshi Eijiro , Itoh Koji , Hayashi Kyozo
Published: October 2006

Abstract: A sensitive two‐site enzyme immunoassay system for mouse β nerve growth factor (NGF) was developed, based on the sandwiching of the antigen between anti‐mouse β NGF antibody IgG coated to a polystyrene tube and anti‐mouse β NGF antibody Fab'‐linked β‐d‐galactosidase (β‐d‐galactoside hydrolase, EC 3.2.1.23). This method has the following advantages: (a) the procedures are simple and rapid compared to bioassay or two‐site radioimmunoassay; (b) antibody Fab'‐β‐d‐galactosidase complex is more stable than 125I‐labeled antibody; (c) purified β NGF is detectable at a concentration as low as 10 pg/ml. Our enzyme immunoassay was used to examine the levels of NGF in some tissues of mice. The submaxillary gland contained a high concentration of NGF. However, other tissues, such as the heart, brain, and skeletal muscle, and serum did not contain detectable NGF. These results support recent findings by other investigators that NGF was not found in the organs/tissues other than the submaxillary gland of mice.
Purification and Characterization of the Heat‐Stable Factors Essential for the Conversion of Lignoceric Acid to Cerebronic Acid and Glutamic Acid: Identification of N‐Acetyl‐l‐Aspartic Acid
Authors: Shigematsu Hidenari , Okamura Nobuyuki , Shimeno Hiroshi , Kishimoto Yasuo , Kan Lou‐sing , Fenselau Catherine
Published: October 2006

Abstract: The conversion of lignoceric acid to cerebronic acid, ceramides, cerebrosides, and glutamic acid is catalyzed by a rat brain particulate preparation. The heat‐stable factor, prepared from calf cerebellum, together with the heat‐labile factor, a pyridine nucleotide, and Mg2+ are essential to all of these metabolic pathways. Our previous work showed that the heat‐stable factor is composed of at least two components, HSF‐1 and HSF‐2, and identified HSF‐2 as d‐glucose‐6‐phosphate. In the current investigation, HSF‐1 was further purified and found to be N‐acetyl‐l‐aspartic acid. In addition, it was discovered that a third component, HSF‐3, is also required for heat‐stable factor activity. A reconstituted system composed of N‐acetylaspartic acid, glucose‐6‐phosphate, and HSF‐3 fully replaced the heat‐stable factor essential for the conversion of lignoceric acid to cerebronic acid and glutamic acid. The reconstituted heat‐stable factor did not show the initial time lag always observed with the crude heat‐stable factor.
Dopamine Uptake by Rat Striatal Synaptosomes: Time‐and Temperature‐Dependent Decay and Protection by Dithiothreitol and Dopamine
Authors: Schoemaker Hans , Nickolson Victor J.
Published: October 2006

The uptake of [3H]dopamine (DA) into rat striatal synaptosomes in the presence of a monoamine oxidase inhibitor was studied using a filtration technique. After a 10‐min preincubation period, a fast initial uptake of [3H] DA was seen. Uptake reached a maximum after 4 min of incubation. If incubation was continued for more than 7 min, a gradual decrease in synaptosomal [3H]DA levels was found. Uptake was dependent on preincubation time; initial uptake velocity and maximal uptake decreased irreversibly with increasing preincubation periods. Moreover, the capacity of the synaptosomes to retain the [3H]DA during longer incubation times was progressively affected. The decrease in initial uptake activity was due to a decrease in the Vmax of the transport system. Dithiothreitol (2.8 mM) protected synaptosomal uptake activity against deterioration at 37°C. Also, DA itself (10‐7M) stabilized the uptake mechanism if added to the suspension before preincubation was started. Since [3H]DA uptake observed after loading the synaptosomes with labeled DA was similar to the uptake seen if the synaptosomes were not previously loaded with DA, it was concluded that under these conditions synaptosomal DA is completely exchangeable with exogenous substrate. Prolonged storage of the synaptosomes at 0°C also resulted in a time‐dependent decrease in uptake activity (t1/2= 116 min). The addition of unlabeled DA or dithiothreitol to the suspension did not affect instability at 0°C.
Muscimol Potentiation of Acidic Amino Acid Release from Cerebellar Synaptosomes Is Chloride Dependent
Authors: Gallo Vittorio , Aloisi Francesca , Levi Giulio
Published: October 2006

In previous studies we have shown that the depolarization‐induced release of preaccumulated acidic amino acids and newly synthesized glutamate from cerebellar synaptosomal preparations is potentiated by γ‐aminobutyric acid (GABA) agonists through a GABAergic presynaptic mechanism. Here we report a systematic analysis of the ionic requirements of the potentiating effect of muscimol on the high K+‐evoked release of d‐[3H]aspartate. Our studies show that: Ca2+, Na+, and Mg2+ are not required for muscimol to exert its effect; a depolarizing concentration of K+ is a necessary, but not sufficient, condition to observe the presynaptic effect in question; and a minimal Cl‐ concentration (50–70 mM) is also required. A possible model based on these findings is proposed.
Changes in Free Amino Acid Levels in Developing Human Foetal Brain Regions
Authors: Datta Salil C. , Mukherjee Kanai L.
Published: October 2006

The levels of free amino acids were determined in human foetal brain regions during prenatal development. Variation in the distribution of amino acids and their rate of change in five segments of the CNS at different stages of ontogeny was observed. Striking developmental changes were found in the levels of aspartic acid in medulla‐pons and spinal cord, glycine in the spinal cord, γ‐aminobutyric acid in the cerebral cortex, glutamic acid in the cerebral cortex, midbrain, and spinal cord, and taurine in the medulla‐pons and spinal cord. At a late gestational period, glutamic acid was found most abundantly over all the brain regions, whereas the level of taurine was highest at an early gestational stage but not in spinal cord.
Biochemical and Pharmacological Characteristics of Conjugated Catecholamines in the Rat Brain
Authors: Karoum F. , Chuang L.‐W. , Wyatt R. J.
Published: October 2006

Mass‐fragmentographic methods are described that enable the simultaneous measurement of total, free, and conjugated catecholamines in brain tissues. These methods were used to assess the distribution, kinetics, and pharmacological characteristics of total, free, and conjugated catecholamines in the hypothalamus, caudate nucleus, hippocampus, and septum. Conjugated norepi‐nephrine (NE) represents ˜20% of total NE in the hypothalamus, septum, and hippocampus, whereas the percentage is ˜ 50% in the caudate nucleus. The percentages of conjugated dopamine (DA) in these brain areas are consistently less than those of NE (˜13%). Although in the hypothalamus the steady‐state concentrations of total, free, and conjugated NE are over four times higher than those of the corresponding total, free, and conjugated DA, the turnover rates of this DA are comparable with those of the corresponding NE. Further, the ratios of conjugated NE or DA turnover rates to those of the total amines are higher than the corresponding ratios of their steady‐state concentrations. Treatments with pargyline (75 mg/kg, i.p.; rats killed 30 and 60 min later) failed to change the contents of conjugated catecholamines in the hypothalamus and the caudate nucleus significantly. Pharmacological manipulation with a number of proto‐typic drugs revealed that although the assay of conjugated catecholamines might shed additional light on the effects of drugs on central catecholamines, the assessment of total or free amines are on the whole equally informative. In conclusion, a detailed assessment of brain conjugated catecholamines is reported. The information provided, fills a gap in our knowledge that has up to now not been adequately addressed.
Study of Differential Effects of Kainic Acid on Metabolic Rates, Utilizing Exogenous or Endogenous Substrates, in Rat Brain Slices
Authors: Poli A. , Migani P. , Contestabile A. , Barnabei O.
Published: October 2006

Abstract: CO2 production from exogenous glucose of cortical, whole hippocampal, and CA3 region hippo‐campal slices, as well as O2 consumption of whole hippocampal slices, were measured in the presence of different concentrations of kainic acid. A moderate, significant increase of CO2 production was seen only in the CA3 region hippocampal preparation at kainic acid concentrations of 10−4–10−2 M. The O2 consumption, at the expense of endogenous energy stores of whole hippocampal slices, was substantially increased by 10−3M kainic acid when the slices were incubated without exogenous glucose. The effect was partly paralleled by the use of high (50 mM) K+ concentration. Some of the possible factors involved in the differential metabolic responses of brain slices to the action of kainic acid are discussed briefly.
Brain Amino Acids Measured by Intracerebral Dialysis in Portacaval Shunted Rats
Authors: Tossman Ulf , Eriksson Siw , Delin Anders , Hagenfeldt Lars , Law David , Ungerstedt Urban
Published: October 2006

Abstract: Changes in brain amino acid uptake and metabolism have been proposed as a possible etiological factor in hepatic encephalopathy. By use of a brain dialysis technique (a thin tube implanted in the brain of the living animal), the extracellular amino acid concentrations in the striatum of portacaval (PC)‐shunted and shamoperated rats were measured. Leucine, phenylalanine, methionine, and glutamine were increased two‐ to sixfold in the PC‐shunted rats, whilst no changes were seen for GABA, valine, glutamate, or isoleucine, confirming previous reports. Aspartate levels were 350% higher in the PC‐shunted rats, and this rise, as well as that of phenylalanine, was significantly correlated with the lower motor activity observed in the PC‐shunted rats, suggesting a possible importance of these amino acids in the etiology of hepatic encephalopathy. The amino acid concentrations measured in whole blood demonstrated the well‐known pattern of low levels of branched‐chain amino acids and increased concentrations of phenylalanine, glutamine, and histidine.
Calmodulin Inhibition of Brain Membrane Phosphorylation
Authors: Thayer Stanley A. , Lemon Robert H. , Fairhurst Alan S.
Published: October 2006

Abstract: Calmodulin has been found to inhibit the phosphorylation of rat brain membrane proteins of molecular weight 14,900–18,900 in a dose‐dependent manner. This phenomenon was seen under conditions in which calmodulin simultaneously produced a stimulatory effect on the phosphorylation of proteins of molecular weight 51,000 and above. This inhibition required calcium, but was not sensitive to cyclic AMP or increasing ATP concentration and was not due to activation of a phosphatase. These results suggest either that calmodulin induces its inhibitory effects on phosphorylation by an indirect mechanism via a presently unknown pathway, or that in addition to the kinase stimulated by calmodulin, there exists another distinct kinase which is inhibited by calmodulin.
Trifluoperazine Inhibits 45Ca2+ Uptake and Catecholamine Secretion and Synthesis in Adrenal Medullary Cells
Authors: Wada Akihiko , Yanagihara Nobuyuki , Izumi Futoshi , Sakurai Seishi , Kobayashi Hideyuki
Published: October 2006

Abstract: In isolated adrenal medullary cells, carbamyl‐choline and high K+ cause the calcium‐dependent secretion of catecholamines with a simultaneous increase in the synthesis of 14C‐catecholamines from [14C]tyrosine. In these cells, trifluoperazine, a selective antagonist of calmodulin, inhibited both the secretion and synthesis of catecholamines. The stimulatory effect of carbamyl‐choline was inhibited to a greater extent than that of high K+. The inhibitory effect of trifluoperazine on carbamylcholine‐evoked secretion of catecholamines was not overcome by an increase in either carbamylcholine or calcium concentration, showing that inhibition by trifluoperazine occurs by a mechanism distinct from competitive antagonism at the cholinergic receptor and from direct inactivation of calcium channels. Doses of trifluoperazine that inhibited catecholamine secretion and synthesis also inhibited the uptake of radioactive calcium by the cells. These results suggest that trifluoperazine inhibits the secretion and synthesis of catecholamines mainly due to its inhibition of calcium uptake. Trifluoperazine seems to inhibit calcium uptake by uncoupling the linkage between cholinergic receptor stimulation and calcium channel activation.
Effect of α‐Fluoromethylhistidine on the Histamine Content of the Brain of W/Wv Mice Devoid of Mast Cells: Turnover of Brain Histamine
Authors: Maeyama Kazutaka , Watanabe Takehiko , Yamatodani Atsushi , Taguchi Yoshitaka , Kambe Hiroshi , Wada Hiroshi
Published: October 2006

Abstract: In the brains of W/Wv mutant mice that have no mast cells, the histidine decarboxylase (HDC) level is as high as in the brain of congenic normal mice (+/+), but the histamine content is 53% of that of +/+ mice. The effects of a‐fluoromethylhistidine (α‐FMH) on the HDC activity and histamine content of the brain of W/Wr and +/+ mice were examined. In both strains, 30 min after i.p. injection of α‐FMH the HDC activity of the brain had decreased to 10% of that in untreated mice. The histamine content decreased more gradually, and after 6 h about half of the control level remained in +/+ mice, whereas histamine had disappeared almost completely in W/Wv mice. It is concluded that the portion of the histamine content that was depleted by HDC inhibitor in a short time is derived from non‐mast cells, probably neural cells. The half‐life of histamine in the brain of W/Wv mice was estimated from the time‐dependent decrease in the histamine content of the brain after administration of a‐FMH: 48 min in the forebrain, 103 min in the midbrain, and 66 min in the hindbrain.
Isolation and Partial Characterization of Plasmalemma from Quiescent Schwann Cells in Denervated Cat Sciatic Nerve
Authors: Ross Stephen M. , Sabri Mohammad I. , Spencer Peter S.
Published: October 2006

Abstract: Fractions enriched in plasma membranes have been obtained from peripheral nerves enriched 89% in quiescent Schwann cells. Fractions were prepared from the intrafascicular tissue of desheathed distal stumps of cat sciatic nerve 8–10 weeks after transection and suture in the upper thigh. Tissue enriched in Schwann cells was minced, homogenized, and centrifuged to remove nuclei and undispersed tissue. Centrifugation of the resulting supernatant produced a pellet that was osmotically shocked, layered over a discontinuous sucrose gradient, and recentrifuged. Fractions enriched in plasma membrane (PM) markers were pooled, osmotically shocked for 16 h, layered over a second discontinuous sucrose density gradient, and recentrifuged. Membrane fractions (0.6 M:0.85 M and 0.85 M: 1.0 M interfaces) contained a homogeneous population of unilamellar vesicles free of myelin. The 0.85 M fraction was enriched in 5′‐nucleotidase, 2′,3′‐cyclic nucleotide 3′‐phosphohydrolase, and specific [3H]ouabain binding, 4.8–, 3.0–, and 5.7–fold over the crude homogenate, respectively. These fractions also demonstrated low enzyme activities for succinate de‐hydrogenase, lactate dehydrogenase, and glucose‐6–phos‐phatase (9, 13, and 15% of control values, respectively). Protein yield of the PM fraction (0.85 M) was approximately 0.6 mg/g of denervated nerve. This preparation should be suitable to characterize the surface properties of Schwann cells free of neuronal regulation.
Selective Presynaptic Cholinergic Neurotoxicity Following Intrahippocampal AF64A Injection in Rats
Authors: Mantione Charles R. , Zigmond Michael J. , Fisher Abraham , Hanin Israel
Published: October 2006

Abstract: Compound AF64A, ethylcholine mustard aziridinium ion (0.4–8 nmol) was stereotaxically administered into rat dorsal hippocampus, and neurochemical changes were determined 5 days later. AF64A treatment, over an almost 10–fold dose range, resulted in a significant (up to 70%) decline in choline acetyltransferase activity. In the same tissue samples, Na+‐dependent choline transport activity was also lowered, with most decreases ranging between 10 and 50% of controls; however, there was no significant correlation (r= 0.39) between these two parameters. Acetylcholinesterase activity was not affected by AF64A treatment when assayed by either histo‐chemical or enzymatic methods. AF64A reduced acetylcholine levels by 43%, but did not alter norepinephrine content or serotonin uptake. These results demonstrate that AF64A can induce a specific, long‐term reduction of cholinergic presynaptic biochemical markers in rat hippocampus. Thus, AF64A can serve as a useful new tool to study the cholinergic system and as an important agent to help develop animal models representing disorders of central cholinergic hypofunction.
Age‐Related Responses of Skeletal Muscle After Ectopic Innervation, with Particular Reference to 16S Acetylcholinesterase, in Adult Rats
Authors: Maldonado Mafalda , Ramirez Beatriz U. , Ruiz Gloria , Yamuy Jack , Inestrosa Nibaldo C.
Published: October 2006

Abstract: The formation of ectopic junctions between the foreign fibular nerve and the soleus muscle of young (35‐day‐old) and mature (200‐day‐old) adult rats was induced by severing the normal nerve 4 weeks after implanting the foreign nerve. The various molecular forms of ace‐tylcholinesterase (AChE) were studied both at the implanted region and at the original denervated endplates. The velocity of contraction was also studied. In young rats the 16S form was first detected in the ectopic junctions around day 5 after reinnervation; this form rapidly increased during the following weeks, reaching a plateau by day 20. By contrast, in mature rats the appearance of the 16S AChE was dramatically delayed; in fact, it could not be observed before day 80 after reinnervation. (The 16S AChE form appeared at day 20 after reinnervation in the original denervated endplates of young rats; however, at the same time, no effect was observed in mature animals.) The original, slow muscle fibers of the soleus became faster upon reinnervation; this change occurred also much earlier in younger than in mature rats. Our results indicate a loss of plasticity in the skeletal muscle of mature rats. We suggest caution in the use of the ectopic innervation model to study development in mature adult rats.
Specific Receptor‐Mediated Inhibition of Cyclic AMP Synthesis by Dopamine in a Neuroblastoma × Brain Hybrid Cell Line NCB‐20
Authors: Berry‐Kravis Elizabeth , Freedman Stephen B. , Dawson Glyn
Published: October 2006

Abstract: Dopamine and dopamine receptor agonists were found to inhibit adenylate cyclase activity dose‐de‐β ndently in a neuroblastoma × Chinese hamster brain explant hybrid cell line NCB‐20. Apomorphine (with an IC50 value of 10 nM) was the most effective inhibitor, followed by 2‐amino‐6,7‐dihydroxy‐1,2,3,4‐tetrahydro‐naphthaline (ADTN), dopamine, and N‐dipropyldopa‐mine. The inhibition was potently reversed by sulpiride, butaclamol, and flupenthixol in a stereospecific manner, but was unaffected by yohimbine, except at high concentrations. Clonidine also inhibited adenylate cyclase activity in these cells and this was reversed by the α2‐adrenoreceptor antagonist yohimbine, but not by sulpiride. [d‐Ala2, d‐Leu5]Enkephalin inhibited adenylate cyclase activity in NCB‐20 cells at nanomolar concentrations; this was reversed by naloxone. All three inhibitory neurotransmitters were able to reverse the stimulation of cyclic AMP synthesis by serotonin or prostaglandin E1The dopamine receptor that modulates cyclic AMP synthesis in NCB‐20 cells is pharmacologically quite distinct from a high‐affinity spiperone binding site identified in these cells, but shows the pharmacologic specificity of the D2 receptor previously described in mammalian brain.
Binding of [3H]Imipramine to Human Platelet Membranes with Compensation for Saturable Binding to Filters and Its Implication for Binding Studies with Brain Membranes
Authors: Phillips Orla M. , Wood Keith M. , Williams D. Clive
Published: October 2006

Abstract: Apparent specific binding of [3H]imipramine to human platelet membranes at high concentrations of imipramine showed deviation from that expected of a single binding site, a result consistent with a low‐affinity binding site. The deviation was due to displaceable, saturable binding to the glass fibre filters used in the assays. Imipramine, chloripramine, desipramine, and fluoxetine inhibited binding to filters whereas 5‐hydroxytryptamine and ethanol were ineffective. Experimental conditions were developed that eliminated filter binding, allowing assay of high and low‐affinity binding to membranes. Failure to correct for filter binding may lead to overestimation of binding parameters, Bmax and KD for high‐affinity binding to membranes, and may also be misinterpreted as indicating a low‐affinity binding component in both platelet and brain membranes. Low‐affinity binding (KD < 2 μM) of imipramine to human platelet membranes was demonstrated and its significance discussed.
Phosphodiesterase Activities for Cyclic Nucleotides in Nerve Endings from the Bovine Posterior Pituitary Gland
Authors: Vale Marcus R. , Baker Ruth V. , Hope Derek B.
Published: October 2006

Abstract: The cyclic nucleotide phosphodiesterase (PDE) activities were studied in a nerve ending fraction from bovine neural lobes. Most of the activity was particulate and unaffected by calcium. Lineweaver‐Burk plots for this fraction showed negative cooperativity with apparent Km values for cyclic AMP of 11 μM and for cyclic GMP of 4 μM. The soluble activities for both cyclic nucleotides were activated by calcium and inhibited by calmodulin‐binding drugs (trifluoperazine and calmidazolium). The apparent Km values were 50 μM for cyclic AMP and 20 μM for cyclic GMP for the soluble activities. Sucrose density gradients resolved the soluble activities into two peaks. The activity with the higher sedimentation rate (MW 122,000 daltons) hydrolysed both cyclic nucleotides and was calcium‐calmodulin‐dependent. The other peak (MW 47,000 daltons) had a higher affinity for cyclic AMP than for cyclic GMP and was calcium‐independent. Solubilized particulate activities gave two main peaks on the density gradient, both calcium‐independent. One was mainly for cyclic AMP (MW 47,000 daltons) and the other mainly for cyclic GMP (MW 133,000 daltons). The function of PDEs in relation to secretion was discussed.
Regional Distribution of the Histamine Metabolite, tele‐Methylimidazoleacetic Acid, in Rat Brain: Effects of Pargyline and Probenecid
Authors: Khandelwal J. K. , Hough Lindsay B. , Green Jack Peter
Published: October 2006

Abstract: tele‐Methylimidazoleacetic acid (t‐MIAA), a major brain histamine metabolite, was measured in nine rat brain regions by a gas chromatography‐mass spectrometric method that also measures the precursor amine, tele‐methylhistamine (t‐MH). The t‐MIAA concentration of cerebellum, medulla‐pons, midbrain, caudate nucleus, hypothalamus, frontal cortex, hippocampus, and thalamus varied 15‐fold, hypothalamus showing the highest level (2.21 nmol/g) and cerebellum the lowest (0.15 nmol/ g). The concentrations of t‐MIAA and t‐MH were significantly correlated in all regions except midbrain, which had relatively more t‐MIAA. Probenecid did not alter whole‐brain t‐MIAA levels. Treatment with pargyline, an inhibitor of monoamine oxidase, lowered the t‐MIAA levels in all regions.
18B1: An Axon‐Specific Antigen Associated with Many Proteins
Authors: Hawkes Richard , Niday Evelyn , Matus Andrew
Published: October 2006

Abstract: We describe an antigen, 18B1, defined by a monoclonal antibody. Immunoperoxidase staining of brain sections shows that 18B1 is selectively associated with neurofilament‐rich axons. Antibody staining of sodium dodecyl sulphate‐gel blots, on the other hand, shows that 18B1 is associated with a large number of proteins, none of which are structural components of brain neurofilaments. The antigen is sensitive to a variety of proteases but is not degraded by various glycosidases, suggesting that 18B1 is probably an amino acid sequence. Its association with neurofilament‐rich axons together with its absence from neurofilaments themselves suggests that it may be involved in mediating interactions between neurofilaments and the proteins that bear it.
Morphological and Biochemical Changes in Myelin Subfractions of Developing Rats Fed Microbial Lipids
Authors: Salvati S. , de Vergiliis L. Conti , Di Felice M. , de Gier J. , Demel R. A. , Crescenzi G. Serlupi , Tomassi G. , Tagliamonte B.
Published: October 2006

Abstract: Morphological, biochemical, and physicochemical studies of myelin subfractions were undertaken on the progeny of Sprague‐Dawley rats fed diets containing lipids either extracted from yeasts grown on n‐alkanes or from margarine. Myelin subfractions obtained from pooled brain homogenates of littermates by sucrose density gradient centrifugation at 7, 14, and 21 days post‐natally were subjected to electron microscopy, sodium dodecylsulfate polyacrylamide gel electrophoresis and assayed for 2′,3′cyclic nucleotide 3′‐phosphohydrolase activity (CNPase; EC 3.1.4.37). Additionally, surface pressure measurements were made of lipid monolayers derived from myelin subfractions, which were subsequently injected with myelin basic proteins. The myelin subfractions of test animals, when compared with those of controls, show an earlier increase in the specific activity of CNPase, the earlier appearance of low‐molecular‐weight proteins, and an increase in the affinity of basic proteins for lipids derived from the myelin light fraction. This biochemistry suggests the presence of a more mature myelin between 7 and 14 days in the experimental group. The morphological studies, however, do not seem to concur with the biochemical data. The observed changes are discussed in relation to the influence of dietary lipids on myelinogenesis.
Ivermectin Interactions with Benzodiazepine Receptors in Rat Cortex and Cerebellum In Vitro
Authors: Williams Michael , Risley Edwin A.
Published: October 2006

Abstract: The anthelmintic macrolide, ivermectin, enhances the binding of benzodiazepine agonist ([3H]‐diazepam) and antagonist ([3H]β‐carboline ethyl ester) ligands to rat cortical and cerebellar membrane preparations. Enhancement of benzodiazepine agonist binding is partially additive with that of γ‐aminobutyric acid (GABA) and is inhibited by etazolate, bicuculline, and the steroid GABA antagonist R5135. Ivermectin‐stimulated benzodiazepine antagonist binding is enhanced by bicuculline and inhibited by GABA and etazolate. The modulatory effects of bicuculline are chloride‐dependent. The stimulatory effects of ivermectin, while quantitatively different in cortex and cerebellum, are qualitatively similar in both brain regions and are reduced in the presence of chloride. Ivermectin effects on benzodiazepine ligand binding to the benzodiazepine receptor complex and the differences in the effects of GABA, bicuculline, and R5135 on ivermectin‐stimulated agonist and antagonist binding may provide evidence for distinct differences in the recognition sites for the two classes of benzodiazepine receptor ligand and their interactions with other components of the receptor complex.
Characterization of Membrane‐Bound and Soluble Catechol‐O‐Methyltransferase from Human Frontal Cortex
Authors: Jeffery Douglas R. , Roth Jerome A.
Published: October 2006

Abstract: Catechol‐O‐methyltransferase (COMT; E.C. 2.1.1.6) from human frontal cortex occurs in both a soluble and membrane‐bound form. Attempts to solubilize the membrane‐bound transferase by repeated washing or by extraction into solutions of high ionic strength were unsuccessful. The finding that Triton X‐100 was capable of solubilizing membrane‐bound COMT suggested that the membrane‐bound transferase is an integral membrane protein. The membrane‐bound and soluble enzymes did not differ in their requirements for magnesium ions or in their pH‐activity profiles; both enzymes showed an optimum near pH 8.0 when assayed in phosphate buffer. In addition, the two enzymes did not differ in the degree of inhibition caused by CaCl2, both enzymes displaying 65% inhibition at 2.5 mM CaCl2. The competitive inhibitors tropolone and nordihydroguaiaretic acid displayed Ki values for the membrane‐bound transferase five‐ to 10‐fold lower than those observed for the soluble transferase. Solubilization of membrane‐bound COMT in Triton X‐100 resulted in an increase in the apparent Km value of the membrane‐bound transferase for dopamine. The increase in Km appeared to be due to apparent competitive inhibition by Triton X‐100 and reached a limiting value of approximately 80 μM. These results confirm that membrane‐bound COMT is an integral membrane protein that may be structurally distinct from soluble COMT.
Incorporation of P0 Protein into Liposomes: Demonstration of a Two‐Domain Structure by Immunochemical and PAGE Analysis
Authors: Koski Carol L. , Franko Maryellen C. , Hudson C. Sue , Shin Mopn L.
Published: October 2006

Abstract: The amphiphilic nature of P0, the major glycoprotein of peripheral nerve myelin, has been suggested previously. In the present study, purified P0 from human peripheral nerve myelin was incorporated into an artificial lipid bilayer consisting of dimyristoyl lecithin and cholesterol. The liposomes were fractionated on a sucrose gradient. The continued expression of P0 antigenicity by the liposomes was shown by specific complement consumption with a multivalent antiserum against P0 or with an IgM monoclonal antibody. Both antibodies recognized P0 expressed on the surface of peripheral nerve myelin and the P0 liposomes. P0 liposomes and peripheral nerve myelin treated with trypsin lost the surface determinant that reacted with the monoclonal antibody. Analysis of the trypsin‐treated liposomes and peripheral nerve myelin by polyacrylamide gel electrophoresis revealed molecular weights for this protein of 19,500 and 20,500, respectively. Similar treatment of the P0 in the fluid phase resulted in many smaller fragments. These results indicate that P0 consists of two domains, a hydrophilic domain accessible to trypsin digestion and a hydrophobic domain, which is potentially trypsin‐sensitive, but shielded by the lipid bilayer. Binding studies with an anti‐P0 monoclonal antibody and polyacrylamide gel analysis of the lipid‐shielded P0 fragment in liposomes and peripheral nerve myelin suggest that the orientation of the protein in the liposome is similar to that in peripheral nerve myelin.
Distribution of Three Enzymes of γ‐Aminobutyric Acid Metabolism in Monkey Retina
Authors: Pusateri Mary Ellen , Carter Joyce G. , Berger Susamma J. , Lowry Oliver H.
Published: October 2006

Abstract: The distributions of glutamate decarboxylase (EC 4.1.1.15), γ‐aminobutyric acid transaminase (EC 2.6.1.19), and succinate semialdehyde dehydrogenase (EC 1.2.1.24) were determined in monkey retina. The decarboxylase was almost restricted to the inner plexiform layer. The transaminase was also highest in this layer, but activities were 40% as high in the adjacent third of the inner nuclear layer and in the ganglion cell and fiber layers. Succinate semialdehyde dehydrogenase was distributed very differently. Although it also showed a peak of activity in the inner plexiform layer, there was a second equal peak in the photoreceptor inner segment layer and a smaller peak in the outer plexiform layer, regions where both γ‐aminobutyric acid transaminase and glutamate decarboxylase were essentially absent.
Uptake of Magnesium by Chromaffin Granules In Vitro: Role of the Proton Electrochemical Gradient
Authors: Fiedler J. , Daniels A. J.
Published: October 2006

Abstract: The chromaffin granule membrane in vitro is impermeable to protons as well as to Mg2+; however, when granules are incubated in the presence of the proton ionophore carbonyl cyanide p‐trifluoromethoxy‐phenylhydrazone or an inhibitor of the granule membrane Mg2+‐dependent ATPase, the metal ion is accumulated inside the granules. This accumulation is dependent upon the granule transmembrane potential. The simultaneous presence of the ATPase inhibitor and the proton ionophore markedly increases metal ion incorporation. Mg2+ incorporation is also promoted by nigericin in the presence of potassium or sodium ions, indicating that Mg2+ accumulation is also dependent upon the transmembrane pH gradient. Concomitant with the Mg2+ accumulation, there is a significant loss of endogenous catecholamines. It is concluded that Mg2+ accumulation is determined by the electrochemical gradient maintained across the membrane. Once the metal ion has accumulated into the granules it displaces catecholamines from their storage sites.
Binding of Tetanus Toxin to Somatic Neural Hybrid Cells with Varying Ganglioside Composition
Authors: Yavin Ephraim , Habig William H.
Published: October 2006

Abstract: 125I‐labelled tetanus toxin interaction with several somatic hybrid cell lines was investigated. Binding of toxin is most effective in NCB‐20, followed by NBr‐10A, NG108‐C15, and SB21‐B1 cells. Specific binding of toxin to NCB‐20 and SB21‐B1 cells is 7– and 60‐fold lower, respectively, in comparison to enriched rat cerebral neuron cultures. The NCB‐20, NBr‐10A, and NG108‐C15 clones display a complex ganglioside pattern, including the presence of [N‐acetyl‐neuraminyl]‐galac‐tosyl ‐ N ‐ acetylgalactosaminyl[N‐ acetylneuraminyl] ‐ galactosylglucosyl ‐ ceramide (GDla) and two unidentified [14C]galactose‐labelled lipid‐soluble compounds, while the SB21‐B1 is most abundant in [N‐acetyl‐neuraminyl]‐galactosylglucosyl ‐ ceramide (GM3) and N ‐ acetyl ‐ galactosaminyl‐[N‐acetyl‐neuraminyl]‐galactosylglucosyl‐ceramide (GM2) gangliosides. None of the cells tested contain measurable levels of [l4C]galactose‐labelled or re‐sorcinol‐positive bands of galactosyl‐N‐ acetyl ‐galacto‐saminyl ‐ [N ‐ acetylneuraminyl ‐ N ‐ acetylneuraminyl]‐galactosylglucosyl‐ceramide (GDlb) and [N‐acetylneuraminyl] ‐ galactosyl ‐ N ‐ acetylgalactosaminyl ‐ [N ‐ acetylneuraminyl ‐ N ‐ acetylneuraminyl] ‐ galactosylglucosyl ‐ceramide (GTlb) gangliosides. After 2 h at 37°C a near plateau of toxin association with NCB‐20 cells is seen. Binding in low‐ionic‐strength medium is 1.35‐fold higher at 37°C than at 4°C, but is reduced by 21 and 51% at 4°C and 37°C, respectively, in physiologic medium. Treatment of NCB‐20 cells with neuraminidase causes a partial loss (29%) of toxin‐binding sites. Binding to the hybrid cells is significantly different from that of cerebral cultures with respect to temperature, salt effect, and sensitivity to neuraminidase, suggesting perhaps a different class of receptors for the toxin. Unlike dibutyryl cyclic AMP or prostaglandin, long‐term serum removal causes a significant increase in toxin binding to NCB‐20 and NG108‐C15 cells. Binding of tQxin to ganglioside‐supplemented SB21‐B1 cells is sevenfold higher compared to untreated cells; it is impaired by neuraminidase added before but not after cell interaction with the toxin at 37°.
Troponin Is Unlikely to Occur in Bovine and Chick Forebrain
Authors: Anthony Frank A. , Babitch Joseph A. , Sharma Satish K.
Published: October 2006

Abstract: A search for the presence of troponin in brain reveals that troponin is below 0.00037% of total bovine brain soluble protein. Troponin levels were examined using G‐actin‐linked Sepharose affinity chromatography and 45Ca binding. The chromatographic and 45Ca binding experiments revealed the presence of several actin and calcium‐binding proteins, none of which corresponded to any troponin subunit. In addition, troponin was not found in any chick brain subfraction analyzed, and the level of troponin in chick nerve ending cytoplasm enriched for troponin was less than 0.023%. Considering that substantial amounts of myosin and actin occur in brain, these findings indicate that troponin is not likely to be a regulator of putative brain actomyosin interactions. The significance of these results and their relation to proposed models for neurotransmitter release is discussed.
Biochemical Abnormalities in Spinal Cord Myelin and CNS Homogenates in Heterozygotes Affected by the Shiverer Mutation
Authors: Cammer Wendy , Kahn Susan , Zimmerman Thomas
Published: October 2006

Abstract: Myelin was purified from the spinal cords of normal mice and mice heterozygous for the shiverer mutation, and measurements were made of the major myelin proteins and lipids and the specific activities of three myelin‐associated enzymes. The myelin purified from the spinal cords of the heterozygotes (shi/+) was deficient by 30–40% in yield and had an apparently unique composition. In particular, when compared with normal mouse spinal cord myelin, there were more high‐molecular‐weight protein, less myelin basic protein, a higher protein‐to‐lipid ratio, and higher specific activities of 2′,3′‐cycIic nucleotide‐3′‐phosphohydrolase (EC 3.1.4.37) and carbonic anhydrase (EC 4.2.1.1) in the myelin purified from the shi/+ animals. These abnormalities were reflected in the composition of shi/+ whole spinal cord, where the protein‐to‐lipid ratio was intermediate between the respective values for +/+ and shil shi spinal cords. Whole brains from shi/+ mice showed deficiencies in galactocerebroside and galactocerebroside sulfate and an increase in total phospholipid, and the lipid composition in the brains of the shi/shi mice was similar to that reported for another dysmyelinating mutant, quaking. The findings provide the first values for the lipids in normal mouse spinal cord myelin and show that heterozygotes are affected by the shiverer mutation. The observations imply that there can be considerable deviation from the normal CNS myelin content and composition without apparent qualitative morphological abnormalities or loss of function and that the amount of myelin basic protein available during myelination may influence the incorporation of other constituents into the myelin membranes. This is also the first report of enrichment in pure normal myelin, over normal spinal cord homogenates, of the enzymes carbonic anhydrase and 5′‐nucleotidase (EC 3.1.3.5).
Presynaptic Nicotinic Cholinergic Receptors Labeled by [3H]Acetylcholine on Catecholamine and Serotonin Axons in Brain
Authors: Schwartz Rochelle D. , Lehmann John , Kellar Kenneth J.
Published: October 2006

Abstract: Nicotinic cholinergic receptor binding sites labeled by [3H]acetylcholine were measured in the cerebral cortices, thalami, striata, and hypothalami of rats lesioned by intraventricular injection of either 6‐hydroxydopamine or 5,7‐dihydroxytryptamine. In addition, [3H]acetylcholine binding sites were measured in the cerebral cortices of rats lesioned by injection of ibotenic acid into the nucleus basalis magnocellularis. [3H]Acetylcholine binding was significantly decreased in the striata and hypothalami of both 6‐hydroxydopamine‐and 5,7‐dihydroxytryptamine‐lesioned rats. There was no change in binding in the cortex or thalamus by either lesion. Ibotenic acid lesions of the nucleus basalis magnocellularis, which projects cholinergic axons to the cortex, did not alter [3H]acetylcholine binding. These results provide evidence for a presynaptic location of nicotinic cholinergic binding sites on catecholamine and serotonin axons in the striatum and hypothalamus.
On the Possible Mechanism of Phenylacetate Neurotoxicity: Inhibition of Choline Acetyltransferase by Phenylacetyl‐CoA
Authors: Potempska Anna , Loo Yen H. , Wisniewski Henry M.
Published: October 2006

Abstract: The influence of phenylacetate, phenylbutyrate, and phenylacetyl‐CoA on the activity of choline acetyltransferase and S‐acetyl‐CoA synthetase was investigated in vitro. Phenylacetyl‐CoA was found to be a very potent inhibitor of choline acetyltransferase, competitive for acetyl‐CoA with Ki of 3.1 × 10−7M. In contrast, millimolar concentrations of phenylacetate and phenylbutyrate were required to inhibit the activity of the enzyme. Activity of S‐acetyl‐CoA synthetase was affected only slightly by the three agents in concentrations of 10−3—‐10−2M. At this time, results are interpreted to suggest that in phenylketonuria, phenylacetate exerts its neurotoxic action through its metabolic product, phenylacetyl‐CoA, which could severely decrease the availability of acetyl‐CoA.
Effects of African Trypanosomiasis on Brain Levels of Dopamine, Serotonin, 5‐Hydroxyindoleacetic Acid, and Homovanillic Acid in the Rabbit
Authors: Stibbs Henry H.
Published: October 2006

Abstract: Serotonin (5‐HT) levels fell by 21% in the mid‐brain‐thalamus‐hypothalamus (MTH) region of the rabbit brain after chronic infection with the protozoan Trypanosoma brucei gambiense. 5‐HT did not decrease significantly in the caudate/putamen (CP) or the pons/medulla (PM) region. 5‐Hydroxyindoleacetic acid (5‐HIAA) levels were unchanged in the MTH and caudate/putamen (CP) but increased by 17% in the pons/medulla (PM) after infection. Dopamine (DA) levels rose by 19% and homovanillic acid (HVA) by 33% in the PM during infection. DA and HVA tended to be lower in the CP of infected rabbits, but the apparent decreases were not statistically significant. DA and HVA levels in the MTH were also unchanged by infection. These neurochemical changes may be involved in the behavioral symptoms that frequently accompany this disease in man and cattle.
Inactivation of Neurotensin by Rat Brain Synaptic Membranes. Cleavage at the Pro10‐Tyr11 Bond by Endopeptidase 24.11 (Enkephalinase) and a Peptidase Different from Proline‐Endopeptidase
Authors: Checler Frédéric , Emson P. C. , Vincent J. P. , Kitabgi P.
Published: October 2006

Abstract: It was shown previously that the tridecapeptide neurotensin is inactivated by rat brain synaptic membranes and that one of the primary inactivating cleavages occurs at the Pro10‐Tyr11 peptide bond, leading to the formation of NT1–10 and NT11–13.The present study was designed to investigate the possibility that this cleavage was catalyzed by proline endopeptidase and/or endopeptidase 24.11 (enkephalinase). Purified rat brain synaptic membranes were found to contain a N‐benzyloxycarbonyl‐Gly‐Pro‐4‐methyl‐coumarinyl‐7‐amide‐hydrolyzing activity that was markedly inhibited (93%) by the proline endopeptidase inhibitor N‐benzyloxycarbonyl‐Pro‐Prolinal and partially blocked (25%) by an antiproline endopeptidase antiserum. In contrast, the cleavage of neurotensin at the Pro10‐Tyr11 bond by synaptic membranes was not affected by N‐benzyloxycarbonyl‐Pro‐Prolinal and the antiserum. When the conversion of NT1–10 to NT1–8 by angiotensin converting enzyme was blocked by captopril and when the processing of NT11–13 by aminopeptidase(s) was inhibited by bestatin, it was found that thiorphan, a potent endopeptidase 24.11 inhibitor, partially decreased the formation of NT1–10 and NT11–13 by synaptic membranes. In conclusion: (1) proline endopeptidase, although it is present in synaptic membranes, is not involved in the cleavage of neurotensin at the Pro10‐Tyr11 bond; (2) endopeptidase 24.11 only partially contributes to this cleavage; (3) there exists in rat brain synaptic membranes a peptidase different from proline endopeptidase and endopeptidase 24.11 that is mainly responsible for inactivating neurotensin by cleaving at the Pro10‐Tyr11 bond.