bluuebunny/crossref_metadata_2025 huggingface dataset:
Localization and Mechanism of Thymidine Transport in the Central Nervous System
Authors: Spector Reynold , Berlinger William G.
Published: October 2006

Abstract: The localization and mechanism of thymidine and deoxyuridine transport in the central nervous system were studied in vivo and in vitro. Previous studies have shown that thymidine enters brain from blood in part via the CSF. In vitro, isolated adult bovine cerebral microvessels, which readily concentrated and phosphorylated deoxyglucose, were unable to concentrate thymidine and deoxyuridine. In vivo, [3H]thymidine (0.2 μM) and [3H]deoxyuridine(0.4 μM) were not extracted more readily than [14C]sucrose in a single pass through the cerebral circulation of rats. In vivo, [3H]thyrnidine retention in CSF and brain after entry from blood was increased when the efflux of [3H]thymidine from CSF and the phosphorylation of [3H]thymidine in brain were depressed by the intraventricular injection of unlabeled thymidine. These studies and previous work suggest that the transfer of thymidine (and deoxyuridine) through the blood‐brain barrier in either direction must be extremely low. The present studies are consistent with the postulate that thymidine is transported by an active transport system in the choroid plexus that transfers thymidine from blood into the CSF; from the CSF, the thymidine enters brain cells and is phosphorylated.
The Benzodiazepine/GABA Receptor Complex: Molecular Size in Brain Synaptic Membranes and in Solution
Authors: Chang Li‐Ren , Barnard Eric A.
Published: October 2006

Abstract: The molecular size of the benzodiazepine (BZ) receptor in the synaptic membrane of brain cortex (bovine or rat) was determined by an improved version of the radiation inactivation method to be 220,000. An identical size was found simultaneously for the associated γ‐aminobutyric acid (GABA) receptor and for the component binding β‐carboline esters. It is proposed that all three activities reside in a single protein or protein complex in the membrane. The size in solution, after extraction into Triton X‐100 medium from exhaustively washed membranes, was estimated by sedimentation constant (9.4S) and by gel filtration (∼230,000 apparent MW), again with the BZ and GABA binding activities behaving identically. This size applies to the component that undergoes photoaffinity labelling by [3H]flunitrazepam in the membrane, and contains a 51,000 Mr polypeptide as the BZ‐binding subunit. It is concluded that a protein complex or oligomer of 200,000–220,000 MW carries a class of BZ‐binding sites and an associated class of GABAA sites.
A Developmental Study of Neuron‐Specific Enolase in Rat Adrenal Medulla
Authors: Vinores Stanley A. , Marangos Paul J.
Published: October 2006

Abstract: The levels of neuron‐specific enolase (NSE) in rat adrenal medulla increase with age. A sharp increase was observed until the age of 15 days. At this time, the NSE level dropped slightly, followed by a gradual increase until the rats were 1 year old. The adrenal medullary NSE levels in males were higher than those observed in females. The difference was seen from 32 days of age, but was not statistically significant until 1 year. This study indicates that NSE can be used as a marker for differentiation in adrenal medulla, as it is used in the central nervous system and in neuroblastoma and pheochromocytoma cells.
Measurement of Methionine‐Enkephalin [Arg6,Phe7] in Rat Brain by Specific Radioimmunoassay Directed at Methionine Sulphoxide Enkephalin[Arg6,Phe7]
Authors: Boarder Michael R. , Lockfeld Alexandre J. , Barchas Jack D.
Published: October 2006

Abstract: A specific and sensitive radioimmunoassay procedure for Metenkephalin[Arg6,Phe7] which allows its measurement in regions of the rat brain is described. The antiserum was raised against the methionine sulphoxide derivative of the peptide, and all samples and standards were oxidized with hydrogen peroxide prior to use in the assay with chloramine T‐oxidized 125I‐labelled Met(O)‐enkephalin[Arg6,Phe7]. The only significant cross‐reactivity was 30% with the reduced heptapeptide Met‐enkephalin[Arg6,Phe7]. The assay showed less than 0.15% cross‐reactivity with fragments of the heptapeptide and with leucine‐enkephalin‐containing peptides. Acid acetone extraction of rat striatum followed by Sephadex G‐50 chromatography and reverse‐phase high pressure liquid chromatography showed that essentially all immunoreactivity co‐chromatographed with Met‐enkephalin[Arg6,Phe7]. This confirmed the specificity of the assay and showed that the striatum does not contain a high concentration of larger molecular weight forms with the heptapeptide at the COOH terminus. Distribution of the heptapeptide followed that of methionine enkephalin, with highest concentrations in the globus pallidus, intermediate levels in caudate‐putamen and hypothalamus, and low levels in cortex and cerebellum.
Evidence that Inhibition of Nicotine‐Mediated Catecholamine Secretion from Adrenal Chromaffin Cells by Enkephalin, β‐Endorphin, Dynorphin (1‐13), and Opiates is not Mediated via Specific Opiate Receptors
Authors: Dean Deanne M. , Lemaire Simon , Livett Bruce G.
Published: October 2006

Abstract: The opioid peptides Met‐ and Leu‐enkephalin, dynorphin (1‐13), and β‐endorphin and the narcotic analgesics, morphine, levorphanol, and dextrorphan all produced a dose‐dependent inhibition of nicotine (5 × 10−6m)‐mediated release of [3H]norepinephrine ([3H]NE) from bovine adrenal chromaffin cells in culture. None of these agents affected [3H]NE release induced by high K+ (56 mm). Although the above results suggest that the opioid peptides and narcotic analgesics inhibit catecholamine release from adrenal chromaffin cells in culture, we suggest that these effects are not mediated by specific opiate binding sites, since (1) the inhibition was only produced with high concentrations of the agents—the threshold concentrations were 10−7 to 10−5m and higher; (2) the inhibition produced by the narcotic analgesics did not display stereospecificity, because the (d‐isomer, dextrorphan, was slightly more active than the l‐isomer, levorphanol; (3) the narcotic antagonists naloxone, naltrexone, and levallorphan did not reverse the inhibition produced by either the narcotic analgesics (e.g., morphine) or the opioid peptides (e.g., dynorphin). These three antagonists themselves inhibited the nicotine‐mediated release of [3H]NE from the adrenal chromaffin cells in culture. Finally (4), the I2‐Tyr1 substituted analogues of β‐endorphin and dynorphin that are biologically less active than the parent compounds produced an inhibition of the nicotine‐mediated [3H]NE release similar to that of their parent compounds. These results do not support the idea that high‐affinity stereospecific opiate binding sites are involved in the inhibitory modulation of nicotinic evoked catecholamine release from bovine adrenal chromaffin cells in culture.
Artifactual Increases in the Concentration of Free GABA in Samples of Human Cerebrospinal Fluid Are Due to Degradation of Homocarnosine
Authors: Grove J. , Schechter P. J. , Tell G. , Rumbach L. , Marescaux C. , Warter J.‐M. , Koch‐Weser J.
Published: October 2006

Abstract: Samples of untreated human cerebrospinal fluid (CSF) were kept at room temperature (20±1°C) up to 72 h, and changes in γ‐aminobutyric acid (GABA) and homocarnosine contents were measured. The concentration of free GABA increased with time, and concomitantly a similar decrease occurred in the concentration of homocarnosine. Total GABA after hydrolysis (present in human CSF at concentrations 40–100 times that of free GABA) did not change. After 2 h the increase in CSF GABA for seven subjects ranged from 42 to 244 pmol/ml. The rate of increase in CSF GABA was positively correlated with the initial homocarnosine concentration. Approximately 5% per h of the initial homocarnosine content was degraded during the first 7 h at room temperature; thereafter the rate gradually decreased. No free GABA was formed in CSF frozen at −70°C for 10 days. When this CSF was restored to room temperature, the formation of free GABA from homocarnosine occurred at essentially the same rate as that observed in fresh CSF. These results demonstrate that the well‐known artifactual increase in GABA concentration of untreated human CSF depends on the concentration of homocarnosine. The rapidity of this increase (up to 2 pmollmlimin) could account for disparities among CSF free GABA concentrations previously reported from normal subjects. It is suggested that measurement of concentrations of total GABA in the CSF would provide a better index of human brain GABA concentration than determination of CSF free GABA.
Regulation of Cyclic AMP Accumulation in a Rat Sympathetic Ganglion: Effects of Vasoactive Intestinal Polypeptide
Authors: Volle Robert L. , Patterson Brian A.
Published: October 2006

Abstract: Cyclic AMP accumulation in rat superior cervical ganglia during synaptic activity occurs by a noncholinergic, nonadrenergic process. Both preganglionic nerve stimulation and 4‐aminopyridine increase ganglion cyclic AMP levels in the presence of atropine or phentolamine. Of the polypeptides tested as putative transmitters, vasoactive intestinal polypeptide (10−6 M) causes ganglion cyclic AMP accumulation comparable to that produced by preganglionic nerve stimulation.
Changes in the Expression of the αα Form of Enolase During Neuroblastoma Differentiation
Authors: Kornblatt M. J. , Keller A. , Legault‐Demare L.
Published: October 2006

Abstract: The relative amounts of the different enolase isozymes present in neuroblastoma cells change during differentiation. When differentiation is induced by low serum in the presence of DMSO (dimethyl sulfoxide), there is a 50% decrease in the concentration of enolase activity associated with the form αα, and an increase in the activity associated with the γ‐containing isozymes (αγ plus γγ); in the absence of DMSO, there is no decrease in αα or in total enolase activity. In order to study the mechanism of the changes in αα, cells differentiated with low serum with and without DMSO were compared. Measurements of the concentration of the α antigen by microcomplement fixation and by immunotitration demonstrate that the decreased enolase activity in DMSO cells is due to a decreased concentration of the α antigen. Measurements of the relative rate of synthesis of the antigen show that the decreased concentration of the α antigen is due to a decreased rate of synthesis. Enolase in differentiated cells is sufficiently stable (t1/2 > 100 h) that a comparison of the relative rates of degradation has not been possible. The decreased synthesis of the α subunit of enolase that occurs under these conditions appears to be a useful model system for studying the de‐expression of the α gene that occurs in vivo during neuronal differentiation.
Pentylenetetrazol May Discriminate Between Different Types of Benzodiazepine Receptors
Authors: Chweh A. Y. , Swinyard E. A. , Wolf H. H.
Published: October 2006

Abstract: The effect of various concentrations of pentylenetetrazol (PTZ) on [3H]flunitrazepam binding to benzodiazepine receptors was investigated by Hofstee and Hill plot analyses. These analyses indicate the presence of two PTZ binding sites in forebrain, whereas a single PTZ binding site is present in cerebellum. The relative proportions of the two PTZ binding sites in forebrain are close to those of benzodiazepine Type II and Type I receptors, respectively. These results suggest that PTZ may actually discriminate between different types of benzodiazepine receptors.
Soluble Gangliosides in Cultured Neurotumor Cells
Authors: Miller‐Podraza Halina , Fishman Peter H.
Published: October 2006

Abstract: The biosynthesis and degradation of glycosphingolipids were studied in cytosolic and membrane fractions obtained from rat glioma C6 cells. Both pools had a similar composition of neutral glycosphingolipids but the soluble pool contained only a few percent of the total. The major ganglioside in C6 cells was GM3, of which only 2% was soluble. Whereas the bulk of the membrane GM3 was accessible to surface labeling procedures, the soluble GM3 was not. Mouse neuroblastoma N18 cells also contained small amounts of cytoplasmic gangliosides corresponding to GM3, GM2, GM1, and GDla. When C6 cells were incubated with medium containing [3H]galactose at 37°C, the specific activity of soluble GM3 initially increased more rapidly than that of membrane GM3; by 4 h, the specific activities in both pools became equal. Total incorporation into the membrane pool, however, was always several‐fold greater even at the shortest incubation times examined. The labeling pattern of neutral glycosphingolipids in both soluble and membrane fractions indicated the existence of a precursor‐product relationship between glucosylceramide and other glycosphingolipids. When labeled cells were transferred to nonradioactive medium, glucosylceramide disappeared the most rapidly, with a 50% loss within <6 h. The turnover rates of other glycosphingolipids were much slower. Although cytosolic GM3 was degraded more rapidly (t1/2= 26 h) than membrane‐bound GM3 (t1/2= 44 h), its turnover rate was much slower than the time required for transport of GM3 to the cell surface (20–30 min). Our results are consistent with the existence of a small intracellular pool of soluble gangliosides and neutral glycosphingolipids that is stable and independent of the main membrane‐bound pool. Although the role of these cytosolic glycolipids is unknown, they do not appear to represent a transport pool between the site of synthesis and the plasma membrane.
Modifications of Ganglioside Patterns in Human Meningiomas
Authors: Berra B. , Riboni L. , De Gasperi R. , Gaini S. M. , Ragnotti G.
Published: October 2006

Abstract: Ganglioside content and distribution were determined in control meninges and in 30 human meningiomas belonging to four different histological types. Irrespective of the histological classification all meningiomas showed a ganglioside content significantly higher than that of control meninges. The analysis of ganglioside distribution in each meningioma showed that in the majority of the cases the increase of ganglioside content was primarily the result of selective accumulation of ganglioside GM3; in the remaining cases ganglioside GM1 was present in a significantly higher amount than in the control dura mater and leptomeninges. A common feature of both types of meningiomas is a simplification of ganglioside pattern, with a shift from the polysialylated to the monosialylated forms. A tentative classification of meningiomas into “GM3‐rich” and “GM1‐rich” types, together with an explanation for the selective accumulation of these two types of ganglioside, is proposed.
Release of Chromaffin Granule Glycoproteins and Proteoglycans from Potassium‐Stimulated PC12 Pheochromocytoma Cells
Authors: Salton Stephen R. J. , Margolis Richard U. , Margolis Renée K.
Published: October 2006

Abstract: Cultured PC12 pheochromocytoma cells were labeled with [3H]gIucosamine, and the glycoproteins and proteoglycans released following potassium‐induced depolarization were fractionated and characterized. Exposure of PC12 cells for 20 min to a high concentration of potassium (51.5 mM in Krebs‐Ringers‐HEPES buffer) results in an approximately sixfold increase in the release of labeled glycoproteins and proteoglycans, compared to incubation in physiological levels of potassium (6 mM). The released complex carbohydrates include chromogranins, dopamine β‐hydroxylase, and two chondroitin sulfate/heparan sulfate proteoglycan fractions, which together account for 7.4% of the soluble cell radioactivity. The chromogranins contained galactosyl(β l ± 3)N‐acetylgalactosamine, as well as several mono‐ and disialyl O‐glycosidically‐linked oligosaccharides, and the tetra‐saccharide AcNeu(α2 ± 3)Gal(β l ± 3)[AcNeu(α2 ± (6)] GalNAcol, obtained by alkaline borohydride treatment of the chromogranin glycopeptides, accounted for almost half of the total chromogranin labeling. The proteoglycan fractions varied in their relative proportions of chondroitin sulfate (23–68%), heparan sulfate (16–23%), and glycoprotein oligosaccharides (16–54%), which are of the triand tetraantennary and O‐glycosidic types. As previously found in the case of proteoglycans from bovine chromaffin granules, the more acidic species has a considerably higher proportion of carbohydrate in the form of sulfated glycosaminoglycans.
Distribution and Properties of Thiol S‐Methyltransferase in Rat Brain
Authors: Hiemke Christoph , Ghraf Rüdiger
Published: October 2006

Abstract: Microsomal thiol S‐methyltransferase (TMT) of rat brain catalysed the methylation of dithiothreitol (Km= 84 μM) and other thiol compounds using S‐adenosylmethionine as methyl donor (Km= 3.7 μM). With increasing polarity of thiol substrates there was a decrease in the maximal velocities of reaction and an increase in the apparent Km values. TMT was found to be unevenly distributed amongst various brain regions, with highest activities in the medulla oblongata and the hippocampus.
UDP‐Galactose:Ceramide Galactosyl Transferase of Isolated Oligodendroglia
Authors: Carruthers Angela , Carey Eric M.
Published: October 2006

Abstract: The activity of UDP‐galactose:ceramide galactosyl transferase (CGalT) has been studied in isolated oligodendroglia from bovine brain white matter and myelinating rat brain. The specific activity and activity per mg DNA are 4‐ and 10‐fold higher in rat oligodendroglia compared with neuronal perikarya from rat brain, and is higher in oligodendroglia from myelinating rat brain compared with bovine oligodendroglia. In membranes isolated from oligodendroglia, the specific activity decreased in the order endoplasmic reticulum > plasma membrane > myelin.
The Structural Conservation of S100 Protein During Evolution: Analysis by Reactivity with a Monoclonal Antibody
Authors: Marks A. , Law J. , Mahony J. B. , Baumal R.
Published: October 2006

Abstract: A hybridoma cell line producing a monoclonal antibody (A4) against bovine S100 protein has been produced by fusing mouse myeloma P3X63/Ag8 cells with spleen cells from a BALB/c mouse immunized with bovine S100 protein. A4 is of the IgG2b subclass and was purified by affinity chromatography on a protein A‐Sepharose column. Brain extracts from several mammalian and one avian species reacted both with polyclonal rabbit anti‐S100 protein antiserum and with A4 in a radio‐immunoassay. Brain extract from dog was a notable exception. It reacted with the rabbit antiserum but not with A4. Therefore A4 reacts with a common epitope that is present on S100 proteins from different vertebrate species but is absent on dog S100 protein.
Circadian Rhythm in Pineal N‐Acetyltransferase Activity: Phase Shifting by Light Pulses (II)
Authors: Binkley Sue
Published: October 2006

Abstract: N‐Acetyltransferase (NAT) is an enzyme whose rhythmic activity in the pineal gland and retina is thought responsible for melatonin circadian rhythms. The enzyme has properties of a circadian biological clock—its rhythm persists in constant conditions and it is precisely controlled by light and dark. Experiments are reported in which light pulses of 1 to 10 h duration were imposed on chicks during their dark‐time. The effect of these pulses upon the NAT was measured and the effect of the pulses on subsequent NAT was also determined. The experiments support the conclusion that the amount and/or duration of dark‐time NAT is limited. This finding is interpreted as supporting the idea that a fixed amount of some substance, an initiator, is synthesized during the subjective day.
Cerebral Polyamine Metabolism: Inhibition of Spermidine Biosynthesis by Dicyclohexylamine
Authors: Porta Raffaele , Camardella Marina , Gentile Vittorio , De Santis Antonio
Published: October 2006

Abstract: Since a specific inhibition of cerebral spermidine (Spd) synthase activity by alicyclic amines was preliminarily observed in vitro, we examined the in vivo inhibitory effectiveness of dicyclohexylamine (DCHA) on Spd biosynthesis in 21‐day‐old rat brain. For this purpose a previously reported HPLC procedure (Porta et al., 1981a) was modified to analyze the cerebral levels of DCHA at the time of polyamine determinations. The intraperitoneally injected DCHA was shown to cross the blood‐brain barrier easily, reaching high levels in the cerebral tissue (∼750 nmol/g brain) within 1 h of its administration. The effect of the drug on the polyamine metabolism resulted in a significant depletion of Spd biosynthesis from the sixth hour after the treatment and in an earlier and prolonged increase of the putrescine (Pt) steady‐state levels. Conversely, the spermine (Spm) endogenous pools remained unchanged throughout the 24‐h post‐DCHA period. Moreover, following the intracerebral administration of [1,4‐14C]Pt, significantly lower specific radioactivity (s.r.a.) values for labeled Pt and Spd were recorded in the brains of DCHA‐treated animals. Conversely, after intracerebral [14C]Spd injection, the s.r.a. of newly formed [14C]Spm remained unchanged, confirming the specificity of the DCHA effect on the Spd biosynthesis.
Antibodies Against the Bovine Brain Glutamate Binding Protein
Authors: Roy Sabita , Michaelis Elias K.
Published: October 2006

Abstract: Antibodies against the purified bovine brain glutamate binding protein (GBP) were raised in rabbits. Both nonderivatized and dinitrobenzene‐derivatized GBP produced strong immunological responses in rabbits. Using the enzyme‐linked immunosorbent assay (ELISA), we have quantified the antibody production and determined the specificity of the antibodies. Bovine brain GBP and the analogous protein from rat brain interacted most strongly with the antibodies. A bacterial glutamate‐aspartate binding protein, as well as the enzymes glutamate dehydrogenase (EC 1.4.1.3), glutamine synthetase (EC 6.3.1.2), and γ‐glutamyl transpeptidase (EC 2.3.2.2), showed little or no cross‐reactivity with the anti‐GBP antibodies. A crude bacterial glutamate decarboxylase (EC 4.1.1.15) preparation gave a small to moderate cross‐reaction with the anti‐GBP antibodies. The sensitivity of the ELISA assay and the specificity of the antibodies were such that GBP levels as low as 3–10 ng could be detected.
Single Doses of Acrylamide Reduce Retrograde Transport Velocity
Authors: Miller Matthew S. , Spencer Peter S.
Published: October 2006

Abstract: Single doses of acrylamide (0–1.3 mmol/kg) produced a dose‐dependent decrease in the transport of 125I‐tetanus toxin to the perikarya of sensory neurons in dorsal root ganglia and motor neurons in ventral spinal cord. Acrylamide was a more potent inhibitor of retrograde transport in sensory axons than in motor axons. Substantially greater doses of N,N′‐methylene‐bis‐acryl‐amide, a reportedly non‐neurotoxic analog of acrylamide, were required to alter the axonal transport of 125I‐tetanus toxin. Velocity of retrograde transport was assessed by determining the position of the leading edge of transported125I‐tetanus toxin at times following single doses of acrylamide. Acrylamide reduced the velocity of 125I‐tetanus toxin transport in a dose‐dependent manner by up to 75%. No change in neuronal uptake of 125I‐tet‐anus toxin was detected. It is concluded that single doses of acrylamide produce profound alterations in retrograde transport which precede the appearance of structural changes in affected nerve fibers.
Formation and Clearance of Norepinephrine Glycol Metabolites in Mouse Brain
Authors: Li Peter P. , Warsh Jerry J. , Godse Damodar D.
Published: October 2006

Abstract: To determine the degree of conversion of 3,4‐dihydroxyphenylethyleneglycol (DHPG) to 3‐methoxy‐4‐hydroxyphenylethyleneglycol (MHPG) and the amount of DHPG eliminated unchanged from the brain, we have examined the kinetics of formation and disappearance of mouse brain MHPG and DHPG following clorgyline (10 mg/kg, i.p.) and/or tropolone (75 mg/kg, i.p.) treatment. During the first 10 min after tropolone, brain DHPG levels accumulated linearly at a rate of 1,300 pmol/g/h, whereas MHPG disappeared exponentially at a rate of 411 pmol/g/h. Following clorgyline administration, brain DHPG declined exponentially at a rate of 1,240 pmol/g/h. In contrast, the elimination of MHPG became a first‐order process only when catechol‐O‐methyltransferase (COMT) was also inhibited in addition to monoamine ox‐idase. Thus, combined clorgyline and tropolone treatment resulted in an exponential decline of MHPG levels at a rate of 524 pmol/g/h, whereas DHPG levels were slightly but significantly elevated compared to control values. When the animals were treated with pargyline (75 mg/kg, i.p.) in combination with clorgyline and tropolone, brain DHPG and MHPG disappeared at rates of 40 and 660 pmol/g/h, respectively. The above observations suggest that mouse brain DHPG is cleared primarily through O‐methylation with minimal direct elimination from brain. Assuming the disposition and clearance of norepinephrine metabolites are similar in mouse and human brain, peripherally measured DHPG in humans is likely derived principally from extracerebral sources and reflects peripheral sympathetic function.
The Relationship Between Arachidonic Acid Release and Catecholamine Secretion from Cultured Bovine Adrenal Chromaffin Cells
Authors: Frye Roy A. , Holz Ronald W.
Published: October 2006

Abstract: Increased arachidonic acid release occurred during activation of catecholamine secretion from cul‐ tured bovine adrenal medullary chromaffin cells. The nicotinic agonist l,l‐dimethyl‐4‐phenylpiperazinium (DMPP) caused an increased release of prcincubated [3H]arachidonic acid over a time course which corre‐ sponded to the stimulation of catecholamine secretion. Like catecholamine secretion, the DMPP‐induced [3H]arachidonic acid release was calcium‐dependent and was blocked by the nicotinic antagonist mecamylamine. Depolarization by elevated K +, which induced catechol amine secretion, also stimulated arachidonic acid release. Because arachidonic acid release from cells probably re‐ sults from phospholipase A2 activity, our findings indicate that phospholipase A2 may be activated in chromaffin cells during secretion.
An Immunological Study of Rat Acetylcholinesterase: Comparison with Acetylcholinesterases from Other Vertebrates
Authors: Marsh David , Grassi Jacques , Vigny Marc , Massoulié Jean
Published: October 2006

Abstract: We have examined the immunoreactivity of acetylcholinesterase from different vertebrate species with a rabbit antiserum raised against the purified rat brain hydrophobic enzyme (G4 form). We found no significant interaction with enzymes from Electrophorus, Torpedo, chicken, and rabbit. The antiserum reacted with acetylcholinesterases from the brains of the other mammalian species studied, with titers decreasing in the following order: rat = mouse > human > bovine. The serum was inhibitory with murine and human acetylcholinesterases, but not with the bovine enzyme. The inhibition was partially depressed in the presence of salt (e.g., l M NaCl). In those species whose acetylcholinesterase was recognized by the antiserum, both soluble and detergent‐soluble fractions behaved in essentially the same manner, interacting with the same antibodies. The apparent immunoprecipitation titer was decreased in the presence of salt, and it did not make any difference whether NaCl was included in the solubilization procedure or added to the extracts. Both G1 and G4 forms of acetylcholinesterase in the soluble and delergent‐soluble fractions were recognized by the antiserum, and in the case of the human enzyme, by monoclonal antibodies produced against human erythrocyte acetylcholinesterase. However, the monomer G1 showed a clear tendency to form smaller complexes and precipitate less readily than the tetramer' G4. Athough we cannot exclude the existence of significant differences belween the various molecular forms of acelylcholinesterase, our results are consistent with the hypothesis that they all derive from the same gene or set of genes by postlranslalional modifications.
Regulation of Nervous System‐Specific S‐100 Protein and Enolase Levels in Adipose Tissue by Catecholamines
Authors: Suzuki Fujiko , Kato Kanefusa , Nakajima Takashi
Published: October 2006

Abstract: The effect of catecholamines on the levels of S‐100 protein and nervous system‐specific enolase (NSE) in epididymal adipose tissue of Wistar rats in vivo was examined by sensitive enzyme immunoassay methods. Soluble S‐100 protein levels in the adipose tissue of 9‐12‐week‐old rats (1.46 ± 0.19 μg/mg protein) were decreased to <50% of those of controls by serial injection (for 4–7 days) of epinephrine (0.1 mg/day) or norepinephrine (0.15 mg) with, however, little effect on the levels of membrane‐bound (pentanol‐extractable) S‐100 protein. A significant decrease in the soluble S‐100 protein levels was observed at 2 h after a single injection of epinephrine (1.04 ± 0.13 μg/mg protein). On the other hand, levels of NSE subunit (γ subunit or 14–3–2 protein) in adipose tissue (0.51 ± 0.03 γγ‐equivalent pmol/mg protein) were increased to 170% of control by serial injection (for 7 days) of epinephrine or norepinephrine with little change of the level of enolase a subunit on a mg protein basis. Isoproterenol had no apparent effect on the levels of soluble S‐100 protein and NSE subunit. These results suggest that the levels of S‐100 protein and NSE in adipose tissue are regulated by catecholamines.
Phorbol Esters Mimic α‐Adrenergic Potentiation of Serotonin N‐Acetyltransferase Induction in the Rat Pineal
Authors: Zatz Martin
Published: October 2006

Abstract: α‐Adrenergic stimulation of the rat pineal gland is known to stimulate phosphatidylinositol turnover and to potentiate the induction of serotonin N‐acetyltransferase (SNAT) activity evoked by submaximal β‐adrenergic stimulation. In some (other) systems tumor‐promoting phorbol esters are known to mimic physiologic stimulation and to enhance specifically the activity of protein kinase C. Here it is shown that phorbol esters specifically mimic the potentiating effect of α‐adrenergic stimulation on SNAT activity in the rat pineal. These effects contribute to the argument for a role for phosphatidylinositol turnover and protein kinase C in mediating α‐adrenergic stimulation.
Improved Method for Isolating Synaptosomes from 11 Regions of One Rat Brain: Electron Microscopic and Biochemical Characterization and Use in the Study of Drug Effects on Nerve Terminal γ‐Aminobutyric Acid in Vivo
Authors: Löscher Wolfgang , Böhme Gerhard , Müller Frank , Pagliusi Sonia
Published: October 2006

Abstract: A procedure is described for the rapid preparation of nerve ending particles (synaptosomes) from 11 regions of one rat brain. The synaptosomal fractions have been characterized by electron microscopy and determination of four marker enzymes, i.e., glutamate decarboxylase (GAD), acetylcholinesterase, succinate dehydrogenase, and glycerol 3‐phosphate dehydrogenase. Comparison with a much lengthier standard (Ficoll‐sucrose) preparation showed that the synaptosomal yield of the new procedure was substantially better as judged by both morphological evaluation and protein recovery. The improved synaptosome preparation was used for determination of regional γ‐aminobutyric acid (GABA) levels in synaptosomal fractions. The postmortem increase in GABA level during removal and dissection of brain tissue and homogenization and fractionation procedures could be minimized by rapid processing of the tissue at low temperatures and inclusion of the GAD inhibitor 3‐mercaptopropionic acid (3‐MP; 1 mM) in the homogenizing medium. The addition of GABA (0.2 mM) to the homogenizing medium did not alter the GAGA levels in the synaptosomes, indicating that no significant redistribution of GABA occurred during subcellular fractionation in sodium‐free media. Synaptosomal GABA levels determined in the 11 rat brain areas showed the same regional distribution as the GABA‐synthesizing enzyme GAD. On the basis of these findings, it was suggested that the synaptosome preparation could be used to evaluate the in vivo effects of drugs on nerve terminal GABA. Treatment of rats with a convulsant dose of 3‐MP (50 mg/kg i.p.) 3 min before decapitation significantly lowered synaptosomal GABA levels in olfactory bulb, hippocampus, thalamus, tectum, and cerebellum. The 3‐MP‐induced seizures and reduction of GABA levels could be prevented by administration of valproic acid (200 mg/kg i.p.) 15 min before the 3‐MP injection. The data indicate that the improved synaptosome preparation offers a convenient method of preparing highly purified synaptosomes from a large number of small tissue samples and can provide useful information on the in vivo effects of drugs on regional GABA levels in nerve terminals.
Depolarisation‐Dependent Protein Phosphorylation and Dephosphorylation in Rat Cortical Synaptosomes Is Modulated by Calcium
Authors: Robinson Phillip J. , Dunkley Peter R.
Published: October 2006

Abstract: The effect of calcium on protein phosphorylation was investigated using intact synaptosomes isolated from rat cerebral cortex and prelabelled with 32Pi. For nondepolarised synaptosomes a group of calcium‐sensitive phosphoproteins were maximally labelled in the presence of 0.1 mM calcium. The phosphorylation of these proteins was slightly decreased in the presence of strontium and absent in the presence of barium, consistent with the decreased ability of these cations to activate calcium‐stimulated protein kinases. Addition of calcium alone to synaptosomes prelabelled in its absence increased phosphorylation of a number of proteins. On depolarisation in the presence of calcium certain of the calcium‐sensitive phosphoproteins were further increased in labelling above nondepolarised levels. These increases were maximal and most sustained after prelabelling at 0.1 mM calcium. On prolonged depolarisation at this calcium concentration a slow decrease in labelling was observed for most phosphoproteins, whereas a greater rate and extent of decrease occurred at higher calcium concentrations. At 2.5 mM calcium a rapid and then a subsequent slow dephosphorylation was observed, indicating two distinct phases of dephosphorylation. Of all the phosphoproteins normally stimulated by depolarisation, only phosphoprotein 59 did not exhibit the rapid phase of dephosphorylation at high calcium concentrations. Replacing calcium with strontium markedly decreased the extent of change observed on depolarisation whereas barium decreased phosphorylation changes even further. Taken together these data suggest that an influx of calcium into synaptosomes initially activates protein phosphorylation, but as the levels of intrasynaptosomal calcium rise protein dephosphorylation predominates. Other phosphoproteins were dephosphorylated immediately on depolarisation in the presence of calcium. The fine control of protein phosphorylation levels exerted by calcium supports the idea that the synaptosomal phosphoproteins could play a role in modulating events such as neurotransmitter release in the nerve terminal.
Involvement of an “Antizyme’ in the Inactivation of Ornithine Decarboxylase
Authors: Laitinen Päivi H.
Published: October 2006

Abstract: DL‐Allylglycine causes a marked increase in mouse brain ornithine decarboxylase (ODC) activity. The amount of immunoreactive enzyme protein increases concomitantly with the activity, but the enzyme protein decreases more slowly than that of the activity. The amount of immunoreactive ODC in brain is many hundred times that of the catalytically active enzyme. The fact that mouse brain cytosol contains high amounts of dissociable antizyme (an inactivating protein) indicates the existence of an inactive, immunoreactive ODC‐antizyme pool. The total antizyme content does not change markedly, but instead there are significant changes in different antizyme pools. Putrescine concentrations start to increase 8 h after treatment with allylglycine and concomitantly with this increase, antizyme is released to inhibit enzyme activity. These results indicate the involvement of antizyme in the inactivation process of ODC.
Study of Dopamine Turnover by Monitoring the Decline of Dopamine Metabolites in Rat CSF After α‐Methyl‐p‐Tyrosine
Authors: Mignot E. , Laude D.
Published: October 2006

Abstract: CSF was continuously withdrawn from the third ventricle of anesthetized rats. CSF 3,4‐dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), and 5‐hydroxyindoleacetic acid concentrations were determined every 15 min by liquid chromatography coupled with electrochemical detection. Acute tyrosine hydroxylase inhibition [with α‐methyl‐p‐tyrosine (α‐MPT)] induced an exponential decline in levels of DOPAC and HVA in CSF. The decline in DOPAC and HVA concentrations was identical in CSF and forebrain but was much slower in the striatum, suggesting that CSF metabolites of 3,4‐dihydroxyphenylethylamine (dopamine) reflect whole forebrain metabolites. The decay in CSF DOPAC and HVA levels after dopamine synthesis inhibition was also used as an in vivo index of forebrain dopamine turnover after various pharmacological treatments. Haloperidol pretreatment accelerated this decay, confirming the increase in brain dopamine turnover induced by neuroleptics. After reserpine pretreatment (15 h before), α‐MPT produced a very sharp decay in levels of DOPAC and HVA. This result indicates that the residual dopamine that cannot be stored after reserpine treatment is very rapidly renewed and metabolized. Nomifensine strongly diminished the slope of DOPAC and HVA level decreases after α‐MPT, a result which can be explained either by a slower dopamine turnover or by the involvement of storage dopamine pools. These results exemplify the use of monitoring the decay of dopamine metabolites after α‐MPT administration in the study of the pharmacological action of drugs on the central nervous system of the rat.
Monoclonal Antibodies to Benzodiazepines
Authors: De Blas Angel L. , Sangameswaran Lakshmi , Haney Steven A. , Park Dongeun , Abraham Carl J. , Rayner Carroll A.
Published: October 2006

Abstract: Four hybridoma lines secreting monoclonal antibodies to benzodiazepines were produced after BALB/c mice were immunized with a benzodiazepine‐bovine serum albumin conjugate. The monoclonal antibodies were purified from ascites fluids, and their binding affinities for benzodiazepines and other benzodiazepine receptor ligands were determined. These antibodies have very high binding affinities for diazepam, flunitrazepam, Ro5‐4864, Ro5‐3453, Ro11–6896, and Ro5–3438 (the KD values are in the 10−9M range). However, these antibodies have low affinities for the benzodiazepine receptor inverse agonists (β‐carbolines) and antagonists (Ro15–1788 and CGS‐8216).
Increased Cerebral Glucose‐6‐Phosphate Dehydrogenase Activity in Alzheimer's Disease May Reflect Oxidative Stress
Authors: Martins Ralph N. , Harper Clive G. , Stokes Gilbert B. , Masters Colin L.
Published: October 2006

Abstract: The activities of the hexose monophosphate pathway enzymes glucose‐6‐phosphate dehydrogenase and 6‐phosphogluconate dehydrogenase were measured at autopsy in control and Alzheimer's disease brains. Enzyme activities did not vary between different areas of brain and were unaltered by age. In Alzheimer's disease, the activities of both enzymes were increased, the glucose‐6‐phosphate dehydrogenase activity being almost double the activity of normal controls. We propose that this increased enzyme activity is a response to elevated brain peroxide metabolism.
Effects of Polyamines on Proline Endopeptidase Activity in Rat Brain
Authors: Soeda Shinji , Yamakawa Naomi , Shimeno Hiroshi , Nagamatsu Atsuo
Published: October 2006

Abstract: The in vitro effects of polyamines on the activity of proline endopeptidase (PEPase) in rat brain cytosol, which contains an endogenous PEPase inhibitor, have been studied. Of the three amines tested (spermine, spermidine, and putrescine), spermine and spermidine markedly enhanced the enzyme activity in brain cytosol. At 6.25 mM spermine or 25 mM spermidine, a 13‐ or 14‐fold enhancement of the enzyme activity was observed. When Mg2+ was used, an approximately fourfold enhancement of the enzyme activity was observed at 50 mM. The enhancement produced by spermine or spermidine was unaffected by Mg2+ up to 50 mM. The activity of purified PEPase was only slightly affected by each polyamine, but it was inhibited 50% by 50 mM Mg2+. On the other hand, 50% inhibition of the enzyme produced by the purified PEPase inhibitor (Mr 7,000; Ki 0.67 mM) was completely restored by addition of 0.7 mM spermine, 3.5 mM spermidine, or 28 mM putrescine. This restoration of inhibition by polyamines was reversed by increasing the inhibitor concentration. These data suggest that polyamines effectively reverse the inhibition of PEPase by its endogenous inhibitor by the reversible formation of a kinetically significant complex. The possible functions of polyamines in the regulation of PEPase in vivo are discussed.
Characterization of the Solubilized A1 Adenosine Receptor from Rat Brain Membranes
Authors: Klotz Karl‐Norbert , Lohse Martin J. , Schwabe Ulrich
Published: October 2006

Abstract: A1 adenosine receptors from rat brain membranes were solubilized with the zwitterionic detergent 3‐[3‐(cholamidopropyl)dimethylammonio]‐1‐propanesulfonate. The solubilized receptors retained all the characteristics of membrane‐bound A1 adenosine receptors. A high and a low agonist affinity state for the radiolabelled agonist (R)‐N6‐[3H]phenylisopropyladenosine(3H]PIA) with KD values of 0.3 and 12 nM, respectively, were detected. High‐affinity agonist binding was regulated by guanine nucleotides. In addition agonist binding was still modulated by divalent cations. The solubilized A1 adenosine receptors could be labelled not only with the agonist [3H]PIA but also with the antagonist 1,3‐diethyl‐8‐[3H]phenylxanthine. Guanine nucleotides did not affect antagonist binding as reported for membrane‐bound receptors. These results suggest that the solubilized receptors are still coupled to the guanine nucleotide binding protein Ni and that all regulatory functions are retained on solubilization.
[3H]Fluphenazine Binding to Brain Membranes: Simultaneous Measurement of D‐1 and D‐2 Receptor Sites
Authors: Morgan David G. , Finch Caleb E.
Published: October 2006

Abstract: [3H]Fluphenazine was used to label both D‐1 and D‐2 dopamine receptors in mouse striatal membranes. The D‐1 and D‐2 specific binding of [3H]fluphenazine was discriminated by the dopamine antagonists SCH‐23390 (D‐1 selective) and spiperone (D‐2 selective). Saturation analyses of these two sites yielded a D‐1 receptor density in mouse striatum of 1,400 fmol/mg of protein and a D‐2 receptor density of 700 fmol/mg of protein. The affinity of [3H]fluphenazine for the D‐2 site was slightly greater than for the D‐1 site; the equilibrium dissociation constant (KD) was 0.7 versus 3.2 nM, respectively. Assay conditions are described that reduce nonspecific binding of [3H]fluphenazine to acceptable levels (35% of total binding at 1 nM [3H]fluphenazine). By comparison of displacement curves from a series of dopaminergic and nondopaminergic ligands, the pharmacological specificity of [3H]fluphenazine binding in mouse striatum was demonstrated to be dopaminergic. Only small amounts of dopamine‐specific (apomorphine‐sensitive) [3H]fluphenazine binding were found in other brain regions. However, chlorpromazine displaced considerable [3H]fluphenazine from all brain regions, including cerebellum, suggesting the presence of a [3H]fluphenazine binding site with a phenothiazine specificity.
Control of Thy‐1 Glycoprotein Expression in Cultures of PC12 Cells
Authors: Doherty Patrick , Walsh Frank S.
Published: October 2006

The effects of nerve growth factor (NGF) and cholera toxin on the expression of the Thy‐1 glycoprotein were examined in cultures of naive and primed PC12 cells using an enzyme‐linked immunoadsorbent assay (ELISA). With primed PC12 cells, NGF induced a rapid increase in Thy‐1 expression over a time course similar to that of neurite regeneration, with half‐maximal and maximal increases apparent at 0.6 and 6 ng/ml NGF. Cholera toxin and dibutyryl cyclic AMP, but not B‐cholera toxin or antibodies to the toxin receptor, were found to inhibit NGF‐induced increases in Thy‐1. Morphological differentiation of naive PC12 cells induced by NGF, but not cholera toxin, was also associated with increased expression of Thy‐1. Despite showing a synergistic effect on morphological differentiation, cholera toxin was again found to inhibit NGF‐induced increases in Thy‐1 expression in cultures of naive PC12 cells. These data suggest that agents that interact directly or indirectly with adenylate cyclase may regulate the responsiveness of PC12 cells to NGF, and as such modulate the expression of the Thy‐1 glycoprotein.
Glucocorticoid Regulation of Glycerol Phosphate Dehydrogenase and Ornithine Decarboxylase Activities in the Spinal Cord of the Rat
Authors: Ortí Eduardo , Moses Daniel F. , Grillo Claudia , De Nicola Alejandro F.
Published: October 2006

Abstract: We examined the effects of glucocorticoids on induction of glycerol phosphate dehydrogenase (GPDH) and ornithine decarboxylase (ODC) in the spinal cord of rats. After a single subcutaneous dose of 5 mg/kg of dexametha‐sone (DEX) phosphate, GPDH activity was maximally increased at 20 h with the effect still persisting for 46 h, in contrast to ODC activity, which was already stimulated at 4 h. The enzyme induction was accompanied by a reduction in number of cytosolic glucocorticoid receptors already at 1 h after DEX treatment, with replenishment at 22 h. A dose‐response curve for DEX demonstrated that the minimal effective dose (0.2 mg/kg) for enzyme induction also reduced the number of cytosolic receptors because of occupation/depletion. The effects were specific for natural and synthetic glucocorticoids, as GPDH and ODC activities were not stimulated by aldosterone, testosterone, estradiol, or progesterone. ODC was induced in the cervical region of the spinal cord as well as in the horse tail plus filum terminale, whereas GPDH responded in the former but not the latter region. Previous work has demonstrated that glucocorticoid receptors are slightly more concentrated in the cervical spinal cord. It is suggested that glucocorticoid induction of these two predominantly glial enzymes occurs by a steroid receptor‐mediated event, as postulated in other regions of the nervous system. In view of the short latency required for induction of ODC, we also examined the effect of inhibitors of transcription and translation. Whereas cycloheximide reduced the stimulatory effect of DEX, a paradoxical stimulation was obtained when DEX and dactinomycin (actinomy‐cin D) were given concomitantly. It is suggested that the inductive responses of GPDH and ODC to glucocorticoids may be different. Considering that GPDH induction has been shown by other laboratories to represent a genomic effect of adrenal steroids, different levels of control may account for the stimulation of ODC in the spinal cord.
Proenkephalin B Messenger RNA in Porcine Tissues: Characterization, Quantification, and Correlation with Opioid Peptides
Authors: Pittius Christoph W. , Kley Nikolai , Loeffler Jean‐Philippe , Höllt Volker
Published: October 2006

Abstract: Concentrations of proenkephalin B (PENK B) mRNA in porcine brain, pituitary, spinal cord, and peripheral tissues were measured using RNA blotting and solution hybridization. A single hybridizing species of ∼2,800 bases in size was present in the CNS, with the highest concentration in the caudate nucleus, followed by hypothalamus and hippocampus. The abundance of PENK B mRNA ranged from 22 pg/μg of poly(A)‐rich RNA in caudate nucleus to <0.1 pg/μg in cerebellum. Concentrations of immunoreac‐tive PENK B‐derived peptides showed a similar distribution, with the exception of the hypothalamus, which had lower PENK B mRNA levels than expected from peptide concentrations. PENK B mRNA of the same size as in the brain was also found in the anterior lobe of the pituitary and in the heart ventricle, whereas in intestine, lung, and kidney, smaller mRNA species of 1,800 bases became apparent by RNA blot analysis. An intermediate size of 2,200 bases was found in heart atrium. As revealed by S1 mapping, however, these smaller mRNAs are not completely homologous with PENK B mRNA, but rather may represent closely related mRNAs from a different gene(s).