bluuebunny/crossref_metadata_2025 huggingface dataset:
Purification and Some Characteristics of an ACTH‐Sensitive Protein Kinase and Its Substrate Protein in Rat Brain Membranes
Authors: Zwiers H. , Schotman P. , Gispen W. H.
Published: October 2006

Abstract: ACTH inhibits the phosphorylation of a rat brain membrane‐bound protein (B‐50). Both the protein kinase and the substrate protein could be extracted from the membranes by means of treatment with Triton X‐100 in 75 mM‐KCl. Using column chromatography over DEAE‐cellulose and ammonium sulphate precipitation a protein fraction (ASP 55–80) enriched in endogenous B‐50 phosphorylating activity was obtained. The time course of the endogenous phosphorylation of B‐50 in this fraction showed a linear incorporation with time for at least 10 min and reached an estimated maximal incorporation of 0.65 mol P/mol B‐50 after 60 min. The inhibition by ACTH1_24 of the B‐50 protein kinase in ASP 55–80 was dose‐dependent; the half‐maximal effective concentration was 5 × 10−6 M, being 10 to 50 times lower as compared with intact synaptic plasma membranes (SPM). cAMP, cGMP and various endor‐phins had no effect on the B‐50 protein kinase. The B‐50 protein kinase required both magnesium and calcium for optimal activity. Using two‐dimensional electrophoresis on polyacrylamide slab gels the B‐50 protein kinase and the B‐50 protein could be identified and purified. The isoelectric point (IEP) of the kinase is 5.5 and the apparent molecular weight 70,000, whereas the IEP of the substrate protein B‐50 is 4.5 and the apparent molecular weight 48,000. Amino acid analysis on microgram quantities of purified kinase and B‐50 protein revealed basic/acidic amino acid ratios in agreement with the respective lEP's. It is speculated that the inhibition of B‐50 protein kinase may be related to known modulatory effects of ACTH and related peptides on certain types of neurotransmission and behaviour.
[3H]Choline Uptake and Metabolism in Nonsynaptic Regions of a Crustacean Sensory Nerve
Authors: Auerbach Anthony , Barker David L.
Published: October 2006

Abstract: The posterior stomach nerve (PSN) is a crustacean sensory nerve containing about 60 cholinergic neurons, which are devoid of synaptic interactions. Kinetic analysis shows that the PSN takes up [3H]choline by both low‐affinity (Km= 163 μM) and high‐affinity (Na+‐dependent) (Km= 1 μM) processes. The capacity of the high‐affinity system is only about 1% that of the low‐affinity system. The high‐affinity system is not tightly coupled to acetylcholine (ACh) synthesis, and it appears that both ACh and phosphorylcholine are formed from an intracellular pool of choline, which is fed by both uptake systems. There are differences in the rates of [3H]choline uptake and 3H metabolite accumulation between regions of the PSN that contain neuronal cell bodies and those that do not. These differences may arise from differences in the relative proportion of neuronal to nonneuronal tissue in each nerve region.
Reduction in Brain Glucose Utilization Rate after Tryptophol (3‐Indole Ethanol) Treatment
Authors: Cornford Eain M. , Crane Paul D. , Braun Leon D. , Bocash William D. , Nyerges Anthony M. , Oldendorf William H.
Published: October 2006

Abstract: 3‐Indole ethanol has been recently identified as the hypnotic agent in trypanosomal sleeping sickness, and because it is formed in vivo after ethanol or disulfiram treatment, it is also associated with the study of alcoholism. When administered intraperitoneally to rats (250 mg/kg) tryptophol induced a sleep‐like state that lasted less than an hour (no righting reflex was apparent 2 min after injection, but it returned at 11 min in bovine serum albumin solution, and 47 min in 40% ethanol solution). In ethanol solutions, tryptophol reduced brain cortical glucose utilization by 55% to the basal brain metabolic rate, and this effect lasted less than 1 h. Synergistic effects of tryptophol and ethanol were suggested by the observation that in albumin solution, tryptophol reduced brain glucose utilization by 35%, but a normal rate was not observed until 2 h postinjection.
Homologous Mammalian Brain Cell Lysate System for the Initiation and Translation of Exogenous mRNAs
Authors: Gilbert Brian E. , Mattick John S.
Published: October 2006

Abstract: The rate of protein synthesis in mammalian brain tissue is affected by a variety of physiological conditions, both natural and induced. The process of initiation may be involved in some of the observed changes, although as yet the actual rates of initiation of natural mRNAs have not been directly measured in these circumstances. One approach to studying the regulation of protein synthesis in brain tissue would be to utilize a homologous cell‐free system to examine in vitro the translation of various added mRNAs. The present report describes a micrococcal nuclease‐treated cell‐free lysate system derived from fetal mouse brain tissue which is capable of actively initiating and translating exogenously added mRNA. Sodium dodecyl sulfate‐polyacrylamide slab gel electrophoretic analysis of the specific protein products of the reaction mixture allowed a qualitative and quantitative assessment of the translational process under a variety of experimental conditions. Optimal conditions for mRNA‐dependent protein synthesis were the following: 30°C incubation temperature; 80–100 mM‐KCl; 2.1 mM‐Mg2+; 50 μM‐spermhe; and 10 μg/ml poly A(+) mRNA. Incorporation of L‐[35S]methionine into proteins required ATP, GTP, and an energy regenerating system. The addition of saturating amounts of a homologous “initiation factors” fraction stimulated incorporation twofold during the first 20 min of incubation, while the patterns of inhibition observed upon the addition of 5 × 10‐5 M‐aurin tricarboxylic acid at various periods during incubation demonstrated the occurrence of multiple rounds of initiation.
Deanol Affects Choline Metabolism in Peripheral Tissues of Mice
Authors: Haubrich D. R. , Gerber N. H. , Pflueger A. B.
Published: October 2006

Abstract: Administration of 2‐dimethylaminoethanol (deanol) to mice induced an increase in both the concentration and the rate of turnover of free choline in blood. Treatment with deanol also caused an increase in the concentration of choline in kidneys, and markedly inhibited the rates of oxidation and phosphorylation of intravenously administered [3H‐methyl]choline. In the liver, deanol inhibited the rate of phosphorylation of [3H‐methyl]choline, but did not inhibit its rate of oxidation or cause an increase in the level of free choline. These findings suggest that deanol increases the choline concentration in blood by inhibition of its metabolism in tissues. Deanol may ultimately produce its central cholinergic effects by inhibition of choline metabolism in peripheral tissues, causing free choline to accumulate in blood, enter the brain, and stimulate cholinergic receptors.
Brain Tubulin Microheterogeneity in the Mouse During Development and Aging
Authors: von Hungen Kern , Chin Rustum C. , Baxter Claude F.
Published: October 2006

Abstract: Mouse brain tubulin was analyzed on isoelectric focusing gels. High‐resolution gels utilizing Bio‐Rad ampholytes (pH 4‐6) revealed 5‐6 bands in the region corresponding to the α‐subunit of tubulin and 10 or more for the β‐subunit. The same general banding pattern was observed regardless of the method of preparation of the tubulin. Two species prominent in the brains of immature mice, α6 and β2, virtually disappeared during maturation, while species β6 to β10 appeared. No significant changes from the mature pattern were seen during aging (examined at 12, 23, and 30 months of age).
The Amino Acid Sequence of the Tryptophan‐Containing Subunit (α‐Subunit) of Bovine Brain S‐100 Protein
Authors: Isobe Toshiaki , Okuyama Tsuneo
Published: October 2006

Abstract: The tryptophan‐containing subunit (α‐subunit) of bovine brain S‐100 protein was purified from a S‐aminoethyl derivative of S‐100a protein, and its amino acid sequence was determined. The α‐subunit contained 93 residues, including one tryptophan, and had a molecular weight of 10,400. The sequence shows an extensive homology (58% identity) to the sequence of another “tryptophan‐free” subunit (β‐subunit) found in both S‐100a and S‐100b protein, and has a calcium binding site characteristic of the “E‐F hand” proteins, such as calmodulin or troponin C. The tryptophan residue is located at position 90 which is presumably adjacent to the C‐terminal end of the α‐helix following the calcium binding loop, and thus appears likely to serve as a specific probe in structure‐function studies of S‐100a protein.
Evidence for Neuronal Localization of Histamine‐N‐Methyltransferase in Rat Brain
Authors: Sperk Günther , Hörtnagl Heide , Reither Harald , Hornykiewicz Oleh
Published: October 2006

Abstract: There is evidence that histamine may be a neurotransmitter in mammalian brain. Histamine in neurons of the central nervous system is easily released and rapidly turned over. The cellular localization of histamine‐N‐methyltransferase, the proposed histamine‐inactivating enzyme, was investigated by measuring its activity in rat striatum after applying neurochemical or electrolytic lesions. The results indicate a major neuronal localization of the enzyme in this area.
Tubulin Synthesis in Rat Forebrain: Studies with Free and Membrane‐Bound Polysomes
Authors: Gilbert Jeffrey M. , Strocchi Paola , Brown Beverly A. , Marotta Charles A.
Published: October 2006

Abstract: Free and membrane‐bound polysomes were prepared from rat forebrain and added to a cell‐free system containing rabbit reticulocyte factors and L‐[35S]methionine. The translation products were analyzed by two‐dimensional gel electrophoresis followed by autoradiography. The free polysomes synthesized actin and at least four major tubulin subunits (α1, α2, β1, and α2) that are found in rat forebrain cytoplasm. The membrane‐bound polysomes synthesized predominantly one protein (MB) in the tubulin region of the two‐dimensional gel. MB has a molecular weight and isoelectric point similar to α‐tubulin. Only trace amounts of α‐ and β‐tubulin and actin were synthesized by the membrane‐bound polysomes. MB co‐purified with cytoplasmic tubulin after two cycles of aggregation and disaggregation. MB synthesized in vitro (from membrane‐bound polysomes) and α‐ and β‐tubulin and actin subunits (synthesized from free polysomes) were digested with Staphylococcus aureus V8 protease, and the resulting peptides were separated by slab gel electrophoresis followed by autoradiography. The peptide pattern of MB was similar but not identical to the peptide patterns of α‐ and β‐tubulin; MB yielded peptides not found in tubulin. We conclude that membrane‐bound polysomes from rat forebrain do not synthesize significant amounts of the predominant tubulin subunits synthesized by free polysomes. A major protein (MB) is synthesized by membrane‐bound polysomes and is similar, but not identical, to α‐tubulin synthesized by free polysomes on the basis of molecular weight, isoelectric point, and peptide analysis.
Rat Brain and Plasma Norepinephrine Glycol Metabolites Determined by Gas Chromatography‐Mass Fragmentography
Authors: Warsh Jerry J. , Godse Damodar D. , Cheung Siu W. , Li Peter P.
Published: October 2006

Abstract: A gas chromatographic‐mass fragmentographic (GC‐MF) procedure is described for the simultaneous quantitation of 3,4‐dihydroxyphenyl‐ethyleneglycol (DHPG) and 3‐methoxy‐4‐hydroxyphenylethyleneglycol (MHPG) in brain tissue and plasma. DHPG and MHPG were assayed as their respective acetyl‐trifluoroacyl esters, using [2H2]DHPG and [2H3]MHPG as internal standards. Assay sensitivities of at least 1 ng per sample were attainable for the quantitation of free glycols, whereas for determination of total DHPG, assay sensitivity was 2.5 ng. Whole rat brain total (99.2 ±4.11 ng/g) and free (13.0 ± 1.14 ng/g) DHPG concentrations were similar to respective total (86.0 ± 3.70 ng/g) and free (12.3 ± 0.41 ng/g) MHPG levels. Total DHPG concentrations exceeded total MHPG levels in hypothalamus (3.0:1), midbrain (1.4:1), pons plus medulla (1.3:1), and hippocampus (1.5:1), whereas in other brain regions the levels of these metabolites were similar. In plasma, however, total DHPG levels were only 20% as high as MHPG concentrations. In mouse brain, DHPG and MHPG occurred almost entirely in free form (>90%), but total DHPG levels were only 50% as high as respective MHPG concentrations. These results emphasize the substantial formation of DHPG compared with MHPG in rat and mouse brain and suggest that DHPG formation and eMux may be of equal or greater importance than MHPG in the metabolic clearance of CNS norepinephrine in some species.
Effects of Fetal Treatment with Methylazoxymethanol acetate at Various Gestational Dates on the Neurochemistry of the Adult Neocortex of the Rat
Authors: Johnston M. V. , Carman A. B. , Coyle J. T.
Published: October 2006

The effects of gestational date (13, 14, 15, and 17 days) of administration of methylazoxymethanol acetate (20 mg/kg) on the cortical synaptic chemistry and morphology of the rat has been examined in adult offspring. Treatment at 13 days of gestation (DG) resulted in cortical hypoplasia that affected primarily the deep layers whereas treatment at 14 and 15 days gestation caused a severe hypoplasia of cortical neurons above layer V and a 66 to 77% reduction in cortical mass. The 17‐DG treatment caused only a 20% reduction in cortical weight with effects apparent only in the superficial layers. At no treatment date did the specific activity of glutamate decarboxylase differ significantly from control. In contrast, presynaptic markers for noradrenergic (tyrosine hydroxylase and norepinephrine) and for serotonergic (serotonin) terminals were increased in concentration in direct proportion to the degree of cortical hypoplasia. The specific activity of choline acetyltransferase was significantly increased at all treatment dates but total activity per cortical slab was significantly reduced on treatment days 13–15.
Induction by Hydrocortisone of Glutamine Synthetase in Mouse Primary Astrocyte Cultures
Authors: Juurlink B. H. J. , Schousboe A. , Jørgensen O. S. , Hertz L.
Published: October 2006

Glutaminc synthetase activity was investigated in developing primary astroglial cultures established from newborn mouse cerebral hemispheres. Between the 2nd and 4th week of culture there was little change in activity under our standard culturing conditions; however, when hydrocortiwne (10 μM) was added to the cultures for 48 h, the enzyme activity increased two‐ to fourfold, depending upon the age of the culture, with maximum response in 2‐week‐old cultures. The addition of dibutyryl cyclic AMP (dRcAMP) to the culture medium caused morphological differentiation of the astroglial cells but eliminated the response of the cells to hydrocortisone. Culturing in elevated serum levels, which delays morphological differentiation and inhibits astroglial cytodifferentiation after exposure to dBcAMP, shifted the time of maximal response to hydrocortisone from 2 to 3 weeks and prevented the abolishment of glutamine synthetase induction by dBcAMP. The induction of glutamine synthetase by hydrocortisone was prevented by actinomycin D (0.5 μg/ml), indicating its dependence upon RNA and protein synthesis. The present work thus confirms reports in the literature that hydrocortisone induces glutamine synthetase in neural tissues, but differs from the findings of Moscona and co‐workers in the chick retina that intact tissues are required for the induction to occur.
Differential Solubilization of γ‐Aminobutyric Acid Receptive Sites from Membranes of Mammalian Brain
Authors: Lester Bruce R. , Miller Ann L. , Peck Ernest J.
Published: October 2006

Sodium‐dependent (+Na) and sodium‐independent (‐Na) receptive sites for γ‐aminobutyric acid (GABA) residing in or on frozen synaptic plasma membranes (SPM) of bovine cerebral cortex were characterized as to binding constants, pharmacologic specificities, and sodium dependence. The SPM fraction was then treated with various concentrations of Triton X‐100 resulting in the loss of pharmacologic specificity, binding characteristics, and sodium dependence associated with +Na GABA receptive sites in SPM. The resulting junctional complex preparation (JC), i.e., a fraction enriched in junctional complexes, possessed only the pharmacologic specificity and binding constants associated with Na receptive sites whether assayed in the presence or absence of 100 mwNaC1. This is probably due to the detergent dispersal or solubilization of the +Na GABA receptive site. The binding constants, KD and Bmax, for –Na GABA binding in SPM were 170 nM and 4.4 pmol/mg protein, while in JC they were 186 nM and 3.7 pmol/mg protein. Under repeated washing the KD was reduced to 60 ± 6.9 nM and the Bmax was reduced to 2.5 ± 0.5 pmol/mg protein in JC, probably owing to the removal of endogenous ligand or inhibitor, and not to inhibition by residual Triton X‐100. Multiple extraction with 0.1% or 0.5% Triton X‐100 did not alter the KB or Bmax, values for the binding of [3H] GABA to JC. Sodium‐independent GABA binding was lost from JC membranes with the use of sodium deoxycholate, probably through solubilization.
Cyclooxygenase Products of Arachidonic Acid Metabolism in Cat Cerebral Cortex After Experimental Concussive Brain Injury
Authors: Ellis Earl F. , Wright Kathy F. , Wei Enoch P. , Kontos Hermes A.
Published: October 2006

Abstract Previous studies have suggested that following experimental fluid percussion brain injury, increased prostaglandin (PG) synthesis, with its concomitant production of oxygen free radicals, causes functional and morphological abnormalities of the cerebral arterioles. The purpose of this study was to chemically determine if PGs are altered following this injury. To facilitate interpretation of neurochemical measurements the cats were ventilated, blood pressure was measured, and a cranial window, for microscopic observation of pial arteriolar diameter was inserted. PG levels were determined in quick‐frozen cortical tissue removed from control and 3 groups of injured cats at 1.5, 8.0, and 60 min after injury. Analysis of PGE2, PGF2α, and 6‐keto‐PGFlα was performed by HPLC and GC/MS. The control levels of PGE2, PGF2α, and 6‐keto‐PGF1α, were 216 ± 44, 210 ± 48, and 48 ± 12 ng/g wet weight, respectively. Following injury, produced by a 22 ms increase in intracranial pressure, the pial arterioles dilated irreversibly and a transient hypertensive response occurred, thereby producing hyperemia. During the maximum hyperemic response, the total PGs were 75% of control. At 8 min after injury, when blood pressure returned to control level, the PGs were 158% of control and PGs fell to 111% of control at 60 min. These experiments supported our previous studies implicating increased PG synthesis in the genesis of the physiologic and morphologic sequelae of experimental concussive brain injury.
Identification and Distribution of Neuroleptic Binding Sites in the Rat Spinal Cord
Authors: Demenge Pierre , Mouchet Patrick , Guerin Bernard , Feuerstein Claude
Published: October 2006

Abstract: Identification of neuroleptic receptor sites in the rat spinal cord could be achieved by the binding of [3H]haloperidol to membranes taken from the different horns. The use of pooled frozen microdiscs punched from these different spinal cord areas allowed the detection of saturable stereospecific binding, as defined in the presence of (+)‐ and (‐)‐butaclamol. Comparison of the binding constants with those obtained in the corpus striatum resulted in similar dissociation constants and Hill's slopes. Maximal binding capacity was quite different, being the greatest in the whole striatum (157 ± 8 fmol/mg protein) followed by the dorsal horn (56 ± 3 fmol/mg protein) and the lateral (34 ± 5 fmol/mg protein) and ventral ones (31 ± 2 fmol/mg protein). The displacement of the labelled ligand by different dopaminergic and nondopaminergic drugs at various concentrations gave similar results in the whole striatum and the spinal cord, giving further support for the existence of a dopaminergic innervation of the spinal cord and showing that dopaminergic receptor sites are distributed through the different spinal horns, with a maximal density in the dorsal horn–as for dopamine levels. No detectable stereospecific binding could be obtained from the surrounding spinal white matter, even at high tissue concentrations. Owing to poor sensitivity of the binding technique, no stereospecific neuroleptic binding could be demonstrated in the whole spinal cord, even at very high tissue concentration, whereas it could be detected in spinal cord tissue sampled from restricted areas of dense dopaminergic innervation.
Electrophoretic Analysis of Axonally Transported Proteins in Toad Retinal Ganglion Cells
Authors: Skene J. H. Pate , Willard Mark
Published: October 2006

Abstract: As a preliminary step to studying changes in axonal transport in regenerating neurons, we have analyzed the composition and organization of polypeptides normally axonally transported in a neuronal system capable of regeneration, i.e., the retinal ganglion cells of the toad, Bufo marinus. We labeled proteins synthesized in the retina with 35S‐methionine and subsequently used one‐dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis to analyze labeled, transported proteins in tissues containing segments of the axons (the optic nerve, optic tract, and optic tecta) of the retinal ganglion cells. The transported polypeptides could be divided into five groups according to their apparent transport velocities. Many of the polypeptides of each group were electrophoretically similar to polypeptides of corresponding groups previously described in rabbit and guinea pig retinal ganglion cells, and in some cases, additional properties of the polypeptides indicated that the transported materials of the two vertebrate classes were homologous. These results serve two purposes. First they establish the retinal ganglion cells of the toad Bufo marinus as a model system in which changes in gene expression related to regeneration may be studied. Second they show that the organization and many aspects of the composition of axonal transport in retinal ganglion cells have been conserved in animals as unrelated as amphibians and mammals.
The Effect of Ouabain on the Release of [14C]Acetylcholine and Other Substances from Synaptosomes
Authors: Vyas S. , Marchbanks R. M.
Published: October 2006

Abstract: The effect of ouabain and dihydroouabain on Na+‐K+ ATPase, 86Rb uptake and the release of [14C]ACh (acetylcholine) from synaptosomal preparations of guinea pigs was compared. At low concentrations of glycoside (<50 μm) there was a good correlation between the potency of ouabain and of dihydroouabain in inhibiting Na+‐K+ ATPase and in causing the release of [l4C]ACh in a nondepolarising medium. Ouabain (200 μM) increased the release of [14C]ACh evoked by 25 mm‐KCl, but not that evoked by 100μm‐veratrine. The enhancement of release was independent of the presence of calcium. It was observed that in addition to [14C]ACh release, choline efflux was also stimulated by ouabain, independently of the presence of Ca2+. Experiments with hemicholinium‐3 showed that the ouabain‐induced increase in choline efflux was not due to an inhibition of reuptake. The effect of ouabain on intrasynaptosomal K+ concentration was measured in order to investigate the degree of depolarisation it caused. The decrease in K+ was found to be similar in magnitude and time course to that caused by veratrine. It was shown that ouabain‐induced depolarisation caused an increased efflux of another positive ion (dibenzyldimethylammonium chloride) and retention of a negatively charged ion (chloride), as would be expected from the operation of the electrochemical potential gradient changing as a result of depolarisation. It is suggested that ouabain acts to stimulate ACh release from synaptosomes as follows: following blockage of the Na+‐K+ ATPase there is rapid depolarisation which, if Ca2+ is present, provokes the normal Ca2+‐dependent transmitter release process to occur. In addition, depolarisation accelerates the leakage of positive ions down their electrochemical potential gradient, but causes a retention of negative ions. Such an action does not depend on the presence of Ca2+, nor is it specific to transmitters.
Pineal and Retinal Serotonin N‐Acetyltransferase Activity: Modulation by Phosphate
Authors: Hamm Heidi E. , Menaker Michael
Published: October 2006

Abstract: Serotonin N‐acetytransferase (NAT) activity in chicken pineal homogenates is increased 16‐fold in the presence of high‐molarity phosphate buffer (0.35 m) as compared with its activity in low‐molarity (0.05 m) phosphate buffer. This phosphate effect on NAT does not depend on ionic, osmotic, or pH changes; rather, it appears to be a direct effect of phosphate on NAT activity. Phosphate also stabilizes NAT activity to thermal inactivation and inactivation caused by incubation at 4°C for 48 h. Stimulation of NAT activity by phosphate occurs only in chick pineal and retina, not in chick cerebrum, cerebellum or liver, nor in rat pineal or other tissues tested. There is a correlation between the occurrence of the phosphate effect and the occurrence of endogenous NAT circadian rhythmicity and light inactivation. The effect of phosphate on NAT activity in homogenates may reflect physiological mechanisms of NAT regulation.
Short‐Term Regulation of Hydroxylase Cofactor Activity in Rat Brain
Authors: Mandell Arnold J. , Russo Patrick V.
Published: October 2006

Abstract: Differential drug effects on hydroxylase cofactor activities were observed in the corpus striatum and the locus coeruleus when conditions of sacrifice were controlled. A conformational stability‐dependent variable degree of stoichiometric coupling between quinonoid dihydropteridine reductase and tyrosine hydroxylase is proposed as a short‐latency influence on hydroxylase cofactor levels.
Membrane Studies in Huntington's Disease: Steady State and Time‐Dependent Fluorescence Spectroscopy of Intact Lymphocytes
Authors: Pettegrew Jay W. , Nichols John S. , Stewart R. Malcolm
Published: October 2006

Abstract: Recent evidence suggests that a membrane abnormality, expressed in peripheral tissues such as the lymphocyte, may be present in Huntington's disease (HD). Both steady state and time‐dependent fluorescence spectroscopic methods were performed on lymphocytes from patients with HD and from age‐ and sex‐matched controls. Lymphocyte membrane dynamics were studied, using fluorescence probes with known specificity for certain membrane areas. These probes included 4‐phenylspiro(furan‐2(3H)‐1′‐phthalan)‐3,3′‐dione (fluorescamine), which binds to surface membrane primary amines, 1–8‐anilinonaphthalene sulfonate (1,8‐ANS), which inserts at the aqueous‐hydrocarbon interface, and 12(9) anthroyl stearate (12(9)AS), which inserts deep in the hydrocarbon core. Steady state fluorescence studies, using fluorescamine, 1–8 ANS, or 12(9)AS, revealed no significant difference between intact HD and control lymphocytes. Time‐dependent energy‐transfer polarization studies for fluorescamine (tryptophan → fluorescamine) did, however, reveal a slower time decay of I1/I for intact HD lymphocytes as compared with controls. This time‐dependent difference may relate to alterations in translational (lateral) and angular mobilities of membrane donors (tryptophan) relative to acceptors (fluorescamine) in intact HD lymphocytes. Such observations support the concept of a membrane abnormality in HD.
In Vivo Formation of 5‐Methoxytryptamine from Melatonin in Rat
Authors: Beck Olof , Jonsson Gösta
Published: October 2006

Abstract: Deacetylation of melatonin to 5‐methoxytryptamine (5‐MT) in vitro and in vivo was investigated in rat liver and brain tissue, using a gas chromatographic‐mass spectrometric 5‐MT assay method. In vitro incubation of liver but not brain (hypothalamic, mesencephalic) slices with melatonin led to a concentration‐dependent formation of small amounts of 5‐MT; the conversion being 0.3–0.8%. In vivo administration of melatonin resulted in a dose‐dependent formation of 5‐MT in small quantities in the liver. The time course showed a peak maximum within 0.5 h, with a rapid decline; the half‐life being about 1 h. 5‐MT could be detected in both the blood and the hypothalamus after in vivo injection of melatonin. The time course of 5‐MT in the blood was similar to that in the liver, but 5‐MT could only be detected in the hypothalamus after large doses shortly after the melatonin injection. MAO had to be inhibited both in the in vitro and in vivo experiments in order to recover 5‐MT, indicating that formed 5‐MT is normally rapidly metabolised by MAO. It is concluded that a small fraction of melatonin can be converted to 5‐MT by deacetylation (by aryl acylamidase) in the liver in vivo, constituting a minor pathway. Such a pathway could not be demonstrated in the brain. Trace amounts of 5‐MT previously reported to be present in various tissues could originate from deacetylation of melatonin in the liver and possibly some other peripheral organs known to contain the deacetylating enzyme. The present results indicate that peripherally formed 5‐MT, a psychoactive compound, is unlikely to have any effect on brain function under normal circumstances.
Biogenic Aldehyde Metabolism in Rat Brain: Subcellular Distribution of Aldose Reductase and Valproate‐Sensitive Aldehyde Reductase
Authors: Turner Anthony J. , Whittle Susan R. , Hryszko John , Guha S. R.
Published: October 2006

Abstract: Reductase activity towards two aldose substrates has been examined in subcellular fractions prepared from rat brain. The reduction of glucuronate, which is sensitive to inhibition by the anticonvulsant drug sodium valproate, corresponds to the major high‐Km aldehyde reductase in brain. Xylose reduction that is insensitive to valproate inhibition has characteristics consistent with the activity of aldose reductase (EC 1.1.1.21). Both enzymes are predominantly localized in the cytosolic fraction. The significance of the location of these two reductases is discussed in relation to the compartmentation of catecholamine metabolism in brain.
Biphasic Effect of Eserine and Other Acetylcholinesterase Inhibitors on the Nicotinic Response to Acetylcholine in Cultured Adrenal Chromaffin Cells
Authors: Mizobe Fumio , Livett Bruce G.
Published: October 2006

Abstract: A pharmacological study was made of the effects of various anticholinesterases (anti‐ChEs) on the release of [3H]noradrenaline ([3H]NA) evoked by acetylcholine (ACh), nicotine, 56 mM K+, and veratridine from bovine adrenal chromaffin cells in culture. The anti‐ChEs chosen were eserine (an inhibitor of both acetylcholinesterase and pseudocholinesterase), 1,5‐bis‐(4‐allyldimethylammoniumphenyl)pentan‐3‐one dibromide (BW284C51) (a specific acetylcholinesterase (AChE) inhibitor), and tetraisopropylprophos‐phoramide (iso‐OMPA) (a specific pseudocholinesterase inhibitor). Acetylcholinesterase (AChE) activity increased in the cells with time in culture beginning at day 4 and reaching a plateau at day 10. In 9–11‐day cultures, both eserine and BW284C51 acted biphasically to increase ACh‐induced [3H]NA release from the cells at concentrations of 10−6M or less whereas higher concentrations reduced or abolished the ACh‐induced release. However, in earlier cultures (days 3–5), when AChE activity of the cells was low, both eserine and BW284C51 produced only a monophasic dose‐dependent inhibition of ACh‐evoked [3H]NA release at high concentrations. When the cells were stimulated with nicotine, an agonist not metabolized by cholinesterase a similar monophasic inhibitory response on the [3H]NA release was elicited by eserine and BW284C51, regardless of the age of the cultured cells. When 56 mM K+ or veratridine was used to depolarize the cells, neither eserine nor BW284C51 affected the [3H]NA release from the cells. Unlike eserine and BW284C51, iso‐OMPA did not enhance ACh‐evoked release in older cultures and at high concentrations (> 10 4M) it produced an inhibition of the [3H]NA release evoked by ACh, nicotine, 56 mM K+, and veratridine. The present results suggest that the stimulatory effect on ACh response by low concentrations of eserine and BW284C51 can be attributed to the protection of ACh against enzymatic hydrolysis, whereas the inhibitory effects produced by higher concentrations of eserine and BW284C51 are thought to be due to an interaction with the nicotinic acetylcholine receptor‐ionophore complex.
Tyrosine Hydroxylase from Bovine Striatum: Catalytic Properties of the Phosphorylated and Nonphosphorylated Forms of the Purified Enzyme
Authors: Lazar Mitchell A. , Lockfeld Alexandre J. , Truscott Roger J. W. , Barchas Jack D.
Published: October 2006

Abstract: The properties of purified tyrosine hydroxylase (TH) from bovine corpus striatum, both native and phosphorylated forms of the enzyme, were studied. TH had a tendency toward greater affinity for tetrahydrobiopterin (BH4) than for the synthetic cofactor 6‐methyltetrahydropterin (6‐MPH4), although the maximal velocity of the TH‐catalyzed reaction was greater with 6‐MPH4. Phosphorylation increased the affinity of TH for cofactor at pH 6.0, with little change in Vmax. At pH 7.0, phosphorylation caused increased activation of TH by increasing Vmax as well as reducing the Km for cofactor. The Km for dopamine was increased twofold by phosphorylation at pH 6.0, but eightfold at pH 7.0. Phosphorylation was not associated with a change in Km for tyrosine at any pH or with any cofactor studied, although the Km for tyrosine of TH was cofactor‐dependent and seven to eight times greater with 6‐MPH4 than with BH4 as cofactor. Heparin and NaCl activated native TH at pH 6.0, but not at pH 7.0. Phosphorylated TH was unaffected by heparin or salt at pH 6.0, but was relatively inhibited at pH 7.0. The data are presented in the context of the physiological environment of TH.
Purification and Chemical Characterization of a W2 Protein from Brain Myelin
Authors: Reig J. A. , Ramos J. M. , Cozar M. , Aguilar J. S. , Criado M. , Monreal J.
Published: October 2006

Abstract: Starting from a pellet of beef brain myelin insoluble in chloroform/ methanol (2:1, vol/vol) (Wolfgram protein fraction), a pure W2 protein with apparent molecular weight of 52,000 was isolated by a simple preparative sodium dodecyl sulfate‐polyacrylamide gel electrophoresis method. A comparative chemical analysis was carried out between purified W2 and a standard tubulin. Glutamic acid and arginine were the N‐terminals detected. Similar peptide maps and amino acid composition were also found in both proteins. Immunological cross‐reactivity was detected when W2 protein was tested against antitubulin serum. These results suggest that W2 protein could have a tubulin‐like protein nature that is associated with the myelin membrane and could play a role in the myelination process.
Agonist Regulation of Muscarinic Acetylcholine Receptors in Rat Spinal Cord
Authors: Taylor John E. , Yaksh Tony L. , Richelson Elliott
Published: October 2006

Abstract: In vitro studies with cultured cells originating from nervous tissue have shown that chronic exposure to muscarinic agonists results in a loss of muscarinic receptors. To determine whether this type of regulation of muscarinic receptor number also occurs in vivo, we infused carbachol into the spinal cords of rats. A single carbachol injection into the lumbar spinal cord caused a significant increase in the nociceptive threshold. This effect of carbachol diminished to control levels after 12 h of repeated agonist injections every 4 h and was blocked by atropine. The desensitization to the antinociceptive effects of carbachol was associated with a loss of muscarinic receptors as determined by the binding of the muscarinic antagonist [3H]quinuclidinyl benzilate. After a 24‐h exposure to carbachol given every 4 h, there was about a 60% loss of binding sites. The loss of muscarinic receptors was also blocked by atropine and was reversible. These results represent direct evidence that a muscarinic agonist can regulate receptor number in the central nervous system and suggest that this loss of receptors is associated with a desensitization to the antinociceptive effects of carbachol injected into the spinal cord.
Postmortem Stability of Enzymes Detoxifying Peroxide In Brain
Authors: Brannan Timothy S. , Maker Howard S. , Bernstein Mitchell
Published: October 2006

Abstract: Glutathione peroxidase, glutathione reductase, and catalase activities were measured to 48 h after death in mouse brains held at temperatures replicating the cooling occurring in human cadaver brain. Glutathione peroxidase was stable for 48 h; catalase was stable for 24 h and then declined 20% in activity. Glutathione reductase was stable for 4 h and then decreased to 55% of its initial activity by 48 h. Perfusion of mouse brain with 0.9% (wt/vol) NaCl did not decrease enzyme activities, indicating that erythrocyte contamination has little effect on measured brain activities. The results suggest that glutathione peroxidase would not be affected by moderate time delays in obtaining human postmortem brains but catalase activity may be affected if brains are not promptly removed. Glutathione reductase is not stable and measurements would require controls carefully matched for postmortem conditions.
A Comparison of 2‐Hydroxyestradiol and U‐0521 (3′4′‐Dihydroxy‐2‐methylpropiophenone, Upjohn) as In Situ and In Vitro Inhibitors of Tyro sine Hydroxylase
Authors: Lloyd Tom , Boyd Brenda , Walega Michael A. , Ebersole Barbara Jones , Weisz Judith
Published: October 2006

Abstract: Feedback inhibition of tyrosine hydroxylase by catechols was evaluated using in situ and in vitro enzyme assays. The three catechol compounds used were norepinephrine, 2‐hydroxyestradiol, and 3′4′‐dihydroxy‐2‐methylpropiophenone (U‐0521, Upjohn); representing endogenous catechol‐amines, catechol estrogens, and a synthetic catechol, respectively. The in situ experiments were performed with dissociated retinal cells from rats and with stationary phase adrenergic‐like neuroblastoma cells (N1E‐115). The catechol estrogen, 2‐hydroxyestradiol, resembled the endogenous catecholamines in its potency to inhibit in vitro and in situ tyrosine hydroxylations with IC50 values of 10 μM in vitro and 100 μM in situ. The drug U‐0521, which has been used as an inhibitor of catechol‐O‐methyltransferase (COMT), was also found to be an inhibitor of tyrosine hydroxylase. Further, it was shown to be more potent than the natural catechols, both in vitro and in situ, with IC50 values of 30–600 nM.
Immunochemical Characterization of the Hyaluronic Acid‐Hyaluronectin Interaction
Authors: Delpech Bertrand
Published: October 2006

Abstract: The interaction between hyaluronic acid (HA) and hyaluronectin (HN) was analyzed by gel chromatography and by the effects of HA on the immunological precipitation of HN. This interaction led to formation of larger molecules, as shown by gel permeation. No inhibition of immune precipitation occurred in liquid phase after addition of HA, but the precipitates in unstained gels were rendered transparent, giving the appearance of inhibition. However, after staining of the gels the precipitates appeared normal. Moreover, a non‐linear decrease of the diffusion rate in antibody‐containing gel was observed as a function of HA concentration at HA:HN weight ratios of 0.75 × 10−3 and higher. A faster movement during electrophoresis, depending on the HA:HN ratio, suppressed the precipitation line when tested by electrosyneresis and produced an increase in migration distance when tested by Laurell's electroimmunoassay. These results show that in the immunochemical detection and quantitation of HN by these techniques consideration should always be given to the amount of HA in the samples.
Resolution and Brain Regional Distribution of Cysteine Sulfinate Decarboxylase Isoenzymes from Hog Brain
Authors: Spears Ronald M. , Martin David L.
Published: October 2006

Abstract: Cysteine sulfinate decarboxylase (CSD; EC 4.1.1.29) activity from porcine brain was resolved into three peaks by hydroxylapatite chromatography. The first two peaks (I and II) did not decarboxylate and were not inhibited by glutamate. The third peak (III) cochromatographed with glutamate decarboxylase (GAD; EC 4.1.1.15) activity. The Km values of cysteine sulfinate for peaks I, II, and III were 5.5 × 10−4m, 1.3 × 10−4m, and 4.5 × 10−3m, respectively. The possibility that the same enzyme was responsible for peak III CSD and GAD activities was suggested by several findings: (1) Mutual competitive inhibition was observed between glutamate and cysteine sulfinate for these activities. (2) Similar first‐order heat‐inactivation curves were obtained for peak III CSD and GAD when incubated at 55xBOC. (3) Both activities were inhibited similarily by ATP and chloride ion. High concentrations of glutamate (0. l m) inhibited peak III CSD activity more than 90% but had no effect on either peak I or II CSD activities. This difference in sensitivity of the isoenzymes to inhibition by glutamate was used to examine the relative regional distributions and the relative contributions to total activity of the glutamate‐sensitive (peak III CSD, GAD) and glutamate‐insensitive (peaks I and II CSD) isoenzymes. Glutamate‐insensitive CSD activity contributed only part of the total activity in all brain regions tested (ranging from 23% in the superior colliculus to 64% in the pons). However, the specific activity of glutamate‐insensitive CSD was more constant than the total or glutamate‐sensitive specific activities among the brain regions tested. The results indicate that GAD is responsible for a significant proportion of the total CSD activity in porcine brain.
Uptake and Calcium‐Dependent Release of Ethylenediamine (1,2‐Diaminoethane) by Rat Brain Slices
Authors: Lloyd H. G. E. , Perkins M. N. , Gaitonde M. K. , Stone T. W.
Published: October 2006

Abstract: The uptake of [14C]ethylenediamine into slices of rat brain and its subsequent evoked release have been studied. An active uptake process was demonstrated by comparing uptake at 37 and 4°C. This uptake showed aKm of 1.36 mM, was partly sodium‐dependent and was reduced by nipecotic acid. Release could be readily evoked by 30 mM potassium, and by electrical stimulation, the release in both cases being calcium‐dependent. In view of these findings and the reported interactions of ethylenediamine with γ‐aminobutyric acid‐related mechanisms, it might be of interest to determine whether this simple diamine occurs endogenously in the mammalian brain.
Tyrosine Hydroxylase Activity in Rat Brain Synaptosomes: Direct Measurement Using High Performance Liquid Chromatography
Authors: Messripour Manoucher , Clark John B.
Published: October 2006

Abstract: By use of high performance liquid chromatography with electrochemical detection to measure dopamine production, tyrosine hydroxylase (EC 1.14.16.2) activity has been measured in rat brain synaptosomes from striatum and forebrain. Normal specific activities three‐ to fivefold higher than previously reported in the literature for radiochemical methods of assay were found. It is suggested that synaptosomes contain a significant amount of endogenous substrate for tyrosine hydroxylase, which causes dilution of the added labelled tyrosine and hence underestimation of the activity of this enzyme when radiochemical methods are used.
Distribution of Cholecystokinin‐Like Peptides in the Human Brain
Authors: Emson P. C. , Rehfeld J. F. , Rossor M. N.
Published: October 2006

Abstract: The regional distribution of cholecystokinin‐like immunoreactivity (CCK‐LI) in postmortem human brain material has been investigated. CCK‐LI was concentrated particularly in the forebrain (all cerebral cortical areas, amygdala, and hippocampus), with smaller amounts in the basal ganglia, hypothalamus, and periventricular grey. Lowest amounts of CCK‐LI were found in the thalamus, cerebellum, and spinal cord.
Lectin‐Reactive Components in White Matter Membranes from Normal and Multiple Sclerosis Brains
Authors: Hukkanen Veijo
Published: October 2006

Abstract: Polypeptides derived from human white matter membranes reacted with the radioiodinated lectins concanavalin A, Lens culinaris phytohemagglutinin, Ricinus communis agglutinin and wheat germ agglutinin after electrophoresis in polyacrylamide pore gradient gels. The molecular weights of these lectin‐reactive bands were estimated by comparison with radioiodinated protein standards by using the linear relationship between log of the molecular weight and log of the gel concentration reached by the protein after electrophoresis in a polyacrylamide gradient gel. The molecular weight estimates for components reactive with concanavalin A were 176,800, 141,200, 72,800, 52,800, 44,700, 40,000, 24,800 and 23,900. The molecular weights of the bands reactive with both wheat germ agglutinin and Lens culinaris phytohemagglutinin were 138,000, 113,500, 92,100, 52,800, 44,700, 24,800 and 23,900. Wheat germ agglutinin was bound also to a band with a molecular weight of 72,800. Ricinus communis agglutinin bound to bands with estimated molecular weights of 138,000, 72,800, 52,800, 44,700, 24,800 and 23,900. The electrophoretic pattern of lectin‐reactive polypeptides derived from normal‐appearing white matter of multiple sclerosis brains was not qualitatively different from the lectin‐binding pattern of control brain membrane polypeptides.
The Effects of Neonatal Pedunclectomy on [3H]Noradrenaline Uptake and the Development of β‐Adrenergic Receptors in the Rat Cerebellum
Authors: Waddington G. , Bower A. J. , Banks P.
Published: October 2006

Abstract: A newly developed method for cutting the cerebellar peduncles in neonatal rats has allowed the study of the development of cerebellar β‐ adrenergic receptors in the absence of noradrenergic afferents. Cutting the cerebellar peduncles of neonatal animals did not affect the pattern of development of the β‐adrenergic receptors, nor their final numbers. Pedunclectomy induced a decline in the ability of slices of cerebellar cortex to accumulate [3H]noradrenaline although high‐affinity noradrenaline uptake was never completely abolished. It is suggested that the remaining high‐affinity noradrenaline uptake cannot be attributed to noradrenergic fibres from the locus coeruleus.
Conformation of Bovine Nerve Root P2 Protein: Characteristics of the Molecule from Circular Dichroism Spectra
Authors: Weise M. J. , Brostoff S. W.
Published: October 2006

Abstract: Circular dichroism (CD) was used to study the conformations of bovine nerve root P2 basic protein, its reduced and carboxymethylated derivative (RCM‐P2), and its large cyanogen bromide fragment (CN1). Data in the far UV show that both the parent protein and RCM‐P2 have conformations dominated by a large amount of β structure. However, the CN1 peptide appears to exist in a largely unordered conformation. Since CN1 lacks short (20 residue) amino‐ and carboxy‐terminal segments of the P2 protein, the spectral data suggest that these regions are important for determining and/or maintaining folding of the P2 protein in aqueous solutions. The P2 protein was found to have a distinctive CD spectrum in the near UV. The characteristics of molar ellipticities indicate that the spectrum contains significant contributions from tyrosine residues, and that these contributions suggest different environments for the two tyrosines in P2 protein. Both environments depend on protein conformation, since CD side chain absorptions are lost when P2 protein is denatured with 5 M urea.