bluuebunny/crossref_metadata_2025 huggingface dataset:
Choline Acetyltransferase Activities in Single Motor Neurons from Vertebrate Spinal Cords
Authors: Kato Takahiko , Murashima Yoshiya L.
Published: December 2006

Abstract: Single cell bodies of spinal motor neurons were isolated from freeze‐dried sections of fresh spinal cords from six species of vertebrates. Single human neurons were also isolated from the spinal cords of three autopsy cases without neurological diseases. Choline acetyltransferase activity of these single neurons was determined by measuring acetyl‐CoA formation from CoASH and acetylcholine by use of the enzymatic amplification reactions, CoA and NADP cyclings. The enzyme activity was unevenly distributed in the cytosol of spinal motor neurons of all species, but not measurable in rabbit dorsal root ganglion cells. The specific activity on a dry weight basis varied widely among the individual neurons from the species studied. The average activity was highest with rat neurons and lowest with yellowtail neurons. The neurons from cold‐blooded animals (bullfrog and yellowtail) had about one‐tenth the activity compared with the warm‐blooded animals (cat, rabbit, rat, and hen). Human neurons, obtained under different morbid and postmortem conditions with three autopsy cases, had very low activities corresponding to those of cold‐blooded animals. Since the choline acetyltransferase activity lost from mouse brain after 11 h at 38°C was 50%, the activity in human neurons was believed to actually be low in vivo.
“Specific” Binding of [3H]Imipramine to Protease‐Sensitive and Protease‐Resistant Sites
Authors: Marcusson J. , Fowler C. J. , Hall H. , Ross S. B. , Winblad B.
Published: December 2006

Abstract: A number of 5‐hydroxytryptamine (5‐HT) uptake inhibitors have been shown to displace the binding of [3H)imipramine to rat cortical membranes in a complex manner with Hill slopes less than unity. Norzimeldine displaced the binding of [3H]imipramine in a biphasic manner with IC50 values for the two components of about 30 nM and 30 μM. This latter site alone was found in tissues that had been treated with a protease. Binding to both of these sites was displaced by 10μM desipramine. The protease‐sensitive [3H]imipramine binding sites were found to be saturable, high‐affinity binding sites with a KD of 8 nM. The number of these sites varied between brain regions and was positively correlated with the regional distribution of [14C]5‐HT but not [3H]noradrenaline uptake. This was not the case however for the protease resistant but desipramine‐displaceable binding sites. Since most previous [3H]imipramine binding studies have been performed with high concentrations of desipramine (10 μ. M) to define “specific binding,” these data would suggest that either protease‐sensitivity or displaceability by 1 μM norzimeldine would give more reliable estimates of the specific binding.
Fluorescence Polarization Analysis, Lipid Composition, and Na+, K+‐ATPase Kinetics of Synaptosomal Membranes in Feline GM1 and GM2 Gangliosidosis
Authors: Wood Philip A. , McBride Michael R. , Baker Henry J. , Christian Samuel T.
Published: December 2006

Abstract: Neurochemical studies were performed on synaptosomal membranes from cats with GM1 or GM2 gangliosidosis to examine possible mechanisms of neuronal dysfunction in these disorders. The basic hypothesis tested was that deficient ganglioside catabolism causes increased ganglioside content of synaptosomal plasma membrane which in turn disrupts normal function. Fluidity characteristics of synaptosomal membranes were examined using fluorescence polarization. Results showed markedly reduced membrane fluidity in both GM1 and GM2 gangliosidosis. These results were supported by a second study which revealed that isolated synaptosomal membranes of GM1 gangliosidosis cats had a 24‐fold increase in total ganglioside content caused predominantly by excess GM1, a 2.3‐fold increased cholesterol content, and a 1.4‐fold increased phospholipid content. Finally, kinetic analysis of synaptosomal plasma membrane Na+, K+‐ATPase from cats with GM1 gangliosidosis showed negligible differences in kinetic parameters compared with controls. Thus, the enzyme appeared protected from the global membrane changes in fluidity and composition. These observations provide evidence for a pathogenetic mechanism of neuronal dysfunction in the gangliosidoses while demonstrating protection of certain vital functional components, such as Na+, K+‐ATPase.
Decline of GABA Uptake in the Hamster Preoptic Area Following Light Offset
Authors: Barkai A. I. , Potegal M. , Kowalik S.
Published: December 2006

Abstract: The uptake of ‐γ‐aminobutyric acid (GABA) was studied in homogenates of the accumbens‐preoptic region (APR) of the hamster brain. Aggressive and non‐aggressive female hamsters, maintained under a reversed 10‐h dark and 14‐h light cycle were sacrificed at various times after light offset. Aliquots of the APR homogenates were incubated with a series of [3H]GABA concentrations to obtain values for Vmax and Km. In both groups Vmax values declined sharply after light offset. The reduction in GABA uptake from early to later dark phase parallels the routine diminution in the aggressive activity of rodents from its peak in the early dark phase; the increase in GABAergic inhibition which presumably results from the reduced uptake may contribute to this change in aggressive activity.
Cyclic AMP Stimulates Serotonin N‐Acetyltransferase Activity in Xenopus Retina In Vitro
Authors: Iuvone P. Michael , Besharse Joseph C.
Published: December 2006

Abstract: The possible involvement of cyclic AMP in the regulation of retinal serotonin N‐acetyltransferase (NAT) activity was investigated using eye cups of Xenopus laevis cultured in a defined medium. Addition of dibutyrylcyclic AMP (dbcAMP) increased retinal NAT activity in eye cups cultured in light. Addition of adenosine or 5′‐AMP under identical conditions was without effect. 3‐Isobutylmethylxanthine (IBMX) increased both retinal cyclic AMP levels and NAT activity in light‐exposed eye cups. Forskolin also increased the concentration of cyclic AMP and the activity of NAT, and the effect of forskolin on both of these parameters was synergistically enhanced by IBMX. The effects of forskolin and of dbcAMP did not require the addition of calcium to the medium; thus, Ca2+‐dependent synaptic transmission does not appear to be required for the response to these drugs. Incubation conditions that activate cyclic AMP‐dependent protein kinase in retinal homogenates had no effect on NAT activity, suggesting that direct phosphorylation of NAT was probably not involved in the response to elevating cyclic AMP in situ. The effect of dbcAMP was blocked by protein synthesis inhibitors. These results suggest that cyclic AMP increases retinal NAT activity by a mechanism that involves protein synthesis, and support a role for cyclic AMP in the nocturnal increase of NAT activity in darkness.
A New Myelin‐Deficient Mutant Hamster: Biochemical and Morphological Studies
Authors: Kunishita Tatsuhide , Tabira Takeshi , Umezawa Hidehiko , Mizutani Makoto , Katsuie Yasutomi
Published: December 2006

Abstract: Biochemical and morphological studies were done on a new trembling mutant hamster CBB. The yield of myelin from the mutant was 30 and 40% of the control at 46 and 140 days of age, respectively, but myelin composition and 2′,3′‐cyclic nucleotide‐3′‐phosphohydrolase (CNPase) activity were normal. Morphologically, about 18% of the axons were myelinated in the mutant optic nerve at 46 days of age, in which the myelinated fibers were those with larger diameters (more than 0.6 μm), while the control had a peak at 0.4 μm in diameter. The ultrastructure and thickness of compact myelin lamellae in the mutant were normal. Myelination and the structure of peripheral nerve myelin appeared normal. The results indicate that the essential defect is the delay and arrest of myelination in the CNS, which is probably caused by either a decreased rate of synthesis of myelin components in oligodendrocytes or a defect in the oligodendrocyteaxon recognition in smaller axons.
Immobilization Decreases Amino Acid Concentrations in Plasma but Maintains or Increases Them in Brain
Authors: Kennett G. A. , Curzon G. , Hunt A. , Patel A. J.
Published: December 2006

Abstract: Immobilization for 2 h significantly decreased plasma concentrations of 13 of 16 amino acids assayed, including the transmitter amine precursors tyrosine and total tryptophan. The level of plasma free tryptophan, however, was increased. Despite the reduced plasma levels, corresponding brain concentrations of many large neutral amino acids (LNAAs) were increased (tryptophan, phenylalanine, valine, leucine, and isoleucine). Brain concentrations of tyrosine and the other amino acids measured were unaltered. The results for the LNAAs were not explained by calculated brain influx rates. Therefore, altered influx kinetics or perhaps altered brain protein metabolism or efflux may be responsible. Comparison of calculated brain influxes and brain concentrations of LNAAs suggests that the rise in level of plasma free tryptophan during immobilization is not responsible for the increase in level of brain tryptophan and that the mechanism responsible for the maintenance of or increase in brain concentrations of the other LNAAs is probably involved. Maintenance of brain concentrations of basic amino acids is explicable by reduced competition for brain uptake.
Studies on Muscarinic Binding Sites in Human Brain Identified with [3H]Pirenzepine
Authors: Lin Siong‐Chi , Olson Kenneth C. , Okazaki Haruo , Richelson Elliott
Published: December 2006

Abstract: Pirenzepine, a potent antimuscarinic agent with apparent selectivity for a subtype (M1) of muscarinic receptors, was used in tritiated form to characterize its binding to human brain tissue. Specific [3H]pirenzepine binding showed rapid association and dissociation. From kinetic and competitive binding experiments, its KD was 5.5 nM and 9 nM, respectively. Regional distribution of [3H]pirenzepine binding determined in parallel with [3H]quinuclidinyl benzilate binding, a nonselective muscarinic antagonist, indicated a significant correlation for the maximum number of binding sites for the two radioligands in 13 brain regions, with the highest amount of binding for each in the putamen and the least in the cerebellum. Binding for [3H]pirenzepine averaged 57% of that for [3H]quinuclidinyl benzilate, with a range of 20% (cerebellum) to 77% (frontal cortex). Most antidepressants and neuroleptics tested had affinities for [3H]pirenzepine binding sites that were not significantly different from their previously reported values obtained with the use of [3H]quinuclidinyl benzilate.
Monoclonal Antibodies to Rat Brain Acetylcholinesterase: Comparative Affinity for Soluble and Membrane‐Associated Enzyme and for Enzyme from Different Vertebrate Species
Authors: Rakonczay Zoltán , Brimijoin Stephen
Published: December 2006

Abstract: Seven unique monoclonal antibodies were generated to rat brain acetylcholinesterase. Upon density gradient ultracentrifugation, immunoglobulin complexes with the monomenc enzyme appeared as single peaks of acetylcholinesterase activity with a sedimentation coefficient approximately 3S greater than that of the free enzyme. This behavior is consistent with the assumption of one binding site per enzyme molecule. Apparent dissociation constants of these antibodies for rat brain acetylcholinesterase calculated on the basis of this assumption ranged from about 10 nA/ to more than 1,000 nM. Some of the antibodies were less able to bind the membrane‐associated enzyme that required detergent for solubilization than the naturally soluble acetylcholinesterase of detergent‐free brain extracts. Species cross‐reactivity was investigated with crude brain extracts from mammals (human, mouse, rabbit, guinea pig, cow, and cat) and from other vertebrates (chicken, frog, and electric eel). Three antibodies bound rat acetylcholinesterase exclusively; one had nearly the same affinity for all mammalian acetylcholinesterases investigated; the remaining three showed irregular binding patterns. None of the antibodies recognized frog and electric eel enzyme. Pooled antibody was found to be suitable for specific immunofluorescence staining of large neurons in the ventral horn of the rat spinal cord and smaller cells in the caudate nucleus. Other potential applications of these antibodies are discussed.
Subcellular Localization of “Peripheral‐Type” Binding Sites for Benzodiazepines in Rat Brain
Authors: Basile Anthony S. , Skolnick Phil
Published: December 2006

Abstract: The binding of [3H]Ro 5–4864, a specific ligand for “peripheral‐type” benzodiazepine binding sites and [3H]Ro 15–1788, a specific ligand for the central benzodiazepine receptors, was determined in subcellular fractions of rat brain. As previously reported, the highest levels of “peripheral‐type” benzodiazepine binding sites and benzodiazepine receptors were found in the crude P1, and P2 fractions, respectively. Purification of these crude fractions revealed that high levels of both [3H]Ro 5–4864 and [3H]Ro 15–1788 binding were present in the mitochondrial and synaptosomal fractions. In contrast, the purified nuclei and myelin contained low levels of both [3H]Ro 5–4864 and [3H]Ro 15–1788 binding.
Oxygen Free‐Radical Reduction of Brain Capillary Rubidium Uptake
Authors: Lo Warren D. , Betz A. Lorris
Published: December 2006

Abstract: Free radicals are proposed to play a role in the injury following cerebral ischemia in which cerebral edema is a prominent feature. To determine whether free radicals might alter the movement of ions and water across the blood‐brain barrier, we examined their effect on brain capillary transport. Rat brain capillaries were isolated, incubated with a system that generates free radicals, and various capillary transport systems were studied. Rubidium uptake was reduced 74% whereas rubidium efflux, glucose transport, and capillary water space were unchanged. The results following the addition of radical scavengers indicated that hydrogen peroxide or a related free radical was the toxic species. These data suggest that free radicals can impair capillary endothelial cell mechanisms that help maintain homeostasis of electrolytes and water in brain.
Effects of Deuterium Substitution on the Catabolism of β‐Phenylethylamine: An In Vivo Study
Authors: Dyck L. E. , Burden D. A. , Boulton A. A.
Published: December 2006

Abstract: β‐Phenylethylamine (PE) hydrochloride injected intraperitoneally into rats was distributed evenly throughout the various regions of rat brain. Similarly, when a mixture of PE and α, α, β, β‐deuterated PE ([2H4]PE) was injected, no regional differences were observed in the ratios of the amounts of [2H4]PE and PE present; however, significantly more [2H4]PE than PE was present, although a 1:1 mixture had been administered. Further experiments in which the amounts of [2H4]PE and PE in whole rat brain, liver, and plasma were quantified confirmed this finding. The maximum [2H4]PE‐to‐PE ratios observed were 67 in whole brain 1 h after injection and 8 in liver and in plasma 45 min after injection. The whole brain [2H4]PE‐to‐PE ratios were decreased by pargyline pretreatment. Subsequent experiments showed that more α, α‐[2H4]PE than PE was present in whole brain, liver, and plasma of rats injected with an equimolar mixture of α, α‐[2H4]PE and PE. In contrast, β, β‐[2H4]PE was not enriched in comparison to PE under the same experimental conditions. We concluded that the basis for the enrichment of [2H4]PE and α, α‐[2H4]PE compared to PE was due to protection of the deuterated analogs from the actions of monoamine oxidase and perhaps aldehyde dehydrogenase; this protection led to pronounced deuterium substitution effects in vivo especially in the brain.
Homology and Diversity Between Intermediate Filament Proteins of Neuronal and Nonneuronal Origin in Goldfish Optic Nerve
Authors: Quitschke Wolfgang , Schechter Nisson
Published: December 2006

Abstract: The predominant intermediate filament proteins of the goldfish optic nerve have molecular weights of 58K. They can be separated into a series of four major isoelectric variants of neuronal (ON1 and ON2) and nonneuronal (ON3 and ON4) origin. The extent of homology between the goldfish 58K intermediate filament proteins themselves and to rat optic nerve vimentin and glial fibrillary acidic protein (GFAP) was investigated. Unlabeled and [32P]orthophosphate‐labeled proteins were subjected to partial hydrolysis by V8 protease, chymotrypsin, and CNBr. The results show that the goldfish intermediate filament proteins share with vimentin and GFAP a 40K chymotrypsin‐resistant core fragment. Phosphorylated moieties appear to be located outside the core region since they are preferentially cleaved off by chymotrypsin and not found associated with the 40K core. In addition, the goldfish ON proteins contain the antigenic site within the core that is common to most intermediate filaments. V8 or CNBr digestion indicates that many fragments that are common to ON1 and ON2 are clearly distinct from fragments that are common to ON3 and ON4. In addition, structural variability is observed between the goldfish intermediate filament proteins and vimentin and GFAP. The results are discussed in terms of intermediate filament structure and their possible role in nerve growth.
Receptor Binding Activities of Biotinylated Derivatives of β‐Nerve Growth Factor
Authors: Rosenberg Michael B. , Hawrot Edward , Breakefield Xandra O.
Published: December 2006

Abstract: β‐nerve growth factor (NGF) was modified by biotinylation via carboxyl group substitution (C‐bio‐NGF) using biotin hydrazide and the coupling reagent 1‐ethyl‐3‐(3‐dimethylaminopropyl)‐carbodiimide, under reaction conditions that yielded an average of 3 biotin additions per NGF subunit. NGF was also biotinylated through amino group substitution, using N‐hydroxysuc‐cinimidyl biotin, to produce derivatives with ratios of one, two, and four biotin moieties per NGF subunit (N‐bio‐NGF). The various biotinylated NGF derivatives were compared with native NGF for their capacity to compete with 125I‐NGF for binding to NGF receptors on rat pheochromocytoma (PC12) cells at 4°C. On the basis of such radioreceptor assays, C‐bio‐NGF was as effective as native NGF in binding to NGF receptors. C‐bio‐NGF was also as effective as native NGF in promoting neunte outgrowth from PC12 cells. In contrast, N‐bio‐NGF containing one biotin per NGF subunit was only 28% as active in binding as native NGF. Increasing the biotin:NGF ratio to 2 or 4 further decreased receptor binding to 13% and 6%, respectively, as compared to native NGF. Once bound to cells, C‐bio‐NGF had the capacity to mediate the specific binding of 125I‐streptavidin to PC12 cells. This binding of streptavidin was prevented by excess native NGF and by antiserum to NGF, but not by RNase A, insulin, cytochrome c, or nonimmune serum. In addition, a variant PC 12 line lacking functional NGF receptors was not labeled by 125I‐streptavidin after prior incubation with C‐bio‐NGF.
Hypothalamic Catecholamine Metabolism in Diabetic Rats: The Effect of Insulin Deficiency and Meal Ingestion
Authors: Glanville N. Theresa , Anderson G. Harvey
Published: December 2006

Abstract: The effect of moderate insulin deficiency of 2 weeks in duration on hypothalamic catecholamine metabolism in food‐deprived and meal‐fed rats was evaluated. Hypothalamic tyrosine content in food‐deprived (from 0700 to 1600 h), diabetic rats was normal. Also normal were the rates of 3,4‐dihydroxyphenylalanine accumulation following aromatic amino acid decarboxylase inhibition, norepinephrine and 3,4‐dihydroxyphenylethylamine (dopamine) clearance after tyrosine hydroxylase inhibition, and intraneuronal amine accumulation following monoamine oxidase inhibition. Differences in hypothalamic amine metabolism were apparent, however, when diabetic and normal rats were fed 2‐g meals. The 3‐methoxy‐4‐hydroxyphenylethyleneglycol sulfate accumulation rate was depressed in diabetic rats by the carbohydrate meal but was stimulated by the tyrosine‐supplemented protein meal. In contrast, the tyrosine‐supplemented diet had no effect on 3, 4‐dihydroxyphenylacetic acid accumulation in diabetic animals, whereas the production rate in normal rats was increased. We conclude that normal responses occurring in hypothalamic, catecholamine metabolism after the consumption of a meal are modified by the presence of diabetes.
Acetoacetate and Glucose as Lipid Precursors and Energy Substrates in Primary Cultures of Astrocytes and Neurons from Mouse Cerebral Cortex
Authors: Lopes‐Cardozo Matthijs , Larsson Orla M. , Schousboe Arne
Published: December 2006

Abstract: Primary cultures of astrocytes and neurons derived from neonatal and embryonic mouse cerebral cortex, respectively, were incubated with [3‐14C]acetoacetate or [2‐14C]glucose. The utilization of glucose and acetoacetate, the production of lactate, d‐3‐hydroxybu‐tyrate, and 14CO2 and the incorporation of 14C and of 3H from 3H2O into lipids and lipid fractions were measured. Both cell types used acetoacetate as an energy substrate and as a lipid precursor; lactate was the major product of glucose metabolism. About 60% of the acetoacetate that was utilized by neurons was oxidized to CO2, whereas this was only ∼20% in the case of cultured astrocytes. This indicates that the rate at which 14C‐labeled Krebs cycle intermediates exchange with pools of unlabeled intermediates is much higher in astrocytes than in neurons. Acetoacetate is a better precursor for the synthesis of fatty acids and cholesterol than glucose, presumably because it can be used directly in the cytosol for these processes; preferential incorporation into cholesterol was not observed in these in vitro systems. We conclude that ketone bodies can be metabolized both by the glial cells and by the neuronal cells of developing mouse brain.
The Lumped Constant in the Deoxyglucose Procedure Declines with Age in Fischer‐344 Rats
Authors: Takei Hidenori , Fredericks Wendy R. , Rapoport Stanley I.
Published: December 2006

Abstract: Concentrations of [14C]2‐deoxy‐D‐glucose ([14C]DG) and of glucose were measured in plasma of arterial and sagittal sinus venous blood from awake Fischer‐344 rats at 3, 12, and 24 months of age, during continuous intravenous infusion of [14C]DG and after a steady‐state arterial plasma concentration of [14C]DG was reached. Brain extraction, i.e., the difference between arterial and venous plasma concentrations divided by the arterial plasma concentration, was calculated for both [14C]DG and glucose. Because exchange of both substances between rat plasma and erythrocytes is slow, the ratio of the brain extraction of [14C]DG to that of glucose is identical to the lumped constant in the deoxyglucose procedure of Sokoloff et al. [J. Neurochem. 28, 897–916, (1977)]. This ratio equaled 0.502 ± 0.015 (SEM) at 3 months, 0.456 ± 0.007 at 12 months, and 0.418 ± 0.006 at 24 months of age (n = 15); the means differed significantly from each other (p < 0.05). The results indicate that the lumped constant declines between 3 and 24 months of age in awake rats, and suggest that many reported age reductions in regional cerebral glucose utilization, of 15–25%, are artifactual.
Increase in the Content of Quinolinic Acid in Cerebrospinal Fluid and Frontal Cortex of Patients with Hepatic Failure
Authors: Moroni F. , Lombardi G. , Carlá V. , Lal S. , Etienne P. , Nair N. P. V.
Published: December 2006

Abstract: Quinolinic acid (QUIN), an excitotoxic tryptophan metabolite, has been identified and measured in human cerebrospinal fluid (CSF) using a mass‐fragmentographic method. Furthermore, its content has been evaluated in frontal cortex obtained at autopsy from the cadavers of patients who died after hepatic coma. During the coma, the concentration of QUIN in the CSF was 152 ± 38 pmol ml‐1. In contrast, the concentration in control patients affected by different pathologies was 22 ± 7 pmol ml‐1. In the frontal cortex of patients who died after episodes of hepatic encephalopathy, the content of QUIN was three times higher than in controls (2.6 ± 0.6 versus 0.80 ± 0.08 nmol/g wet weight). As a result of these investigations we are now able to extend our previous observations on the increase of QUIN in the brains of rats used as experimental models of hepatic encephalopathy to man. QUIN should therefore be added to the list of compounds possibly involved in the pathogenesis and symptomatology of brain disorders associated with liver failure.
Homeostasis of Brain and Cerebrospinal Fluid Calcium Concentrations During Chronic Hypo‐and Hypercalcemia
Authors: Murphy Vincent A. , Smith Quentin R. , Rapoport Stanley I.
Published: December 2006

Abstract: Three‐week‐old rats were made hypocalcemic or hypercalcemic by being fed diets low or high in Ca. Both total and ionized [Ca]s in the plasma decreased about 40% and remained depressed for 4 weeks in rats fed a low‐Ca diet. Plasma [Ca]s in rats fed a high‐Ca diet increased by 30% and remained elevated for 7 weeks. After 8 weeks on the diets, cerebrospinal fluid (CSF) [Ca] changed by <30% whereas brain [Ca] changed by <20% of the chronic changes in plasma ionized [Ca]. Assuming a brain extracellular volume of 20% and noting that brain extracellular volume equilibrates freely with CSF, the findings demonstrate only small perturbations in the Ca content of the brain cellular compartment during sustained hypo or hypercalcemia. Partial regulation of CSF and brain extracellular Ca suggests a role for the blood‐brain barrier in regulating CNS [Ca] during chronic changes in plasma [Ca].
Metaphit Irreversibly Inhibits [3H]threo‐ (±)‐Methylphenidate Binding to Rat Striatal Tissue
Authors: Schweri M. M. , Jacobson A. E. , Lessor R. A. , Rice K. C.
Published: December 2006

Abstract: Metaphit {1‐[1‐ (3‐isothiocyanatophenyl)cyclohexyl]‐piperidine}, a derivative of phencyclidine that contains an isothiocyanate group on the meta position of the aromatic ring, resembles its parent compound (phencyclidine) in its ability to inhibit the binding of the stimulant drug [3H]threo‐ (±)‐methylphenidate to crude synaptosomal membranes from rat Striatal tissue (IC50= 1.4 and 6.2 μM for phencyclidine and Metaphit, respectively). Unlike phencyclidine, however, Metaphit appears to inhibit binding of the radiolabeled stimulant in an irreversible manner, as the degree of inhibition of binding of the stimulant does not diminish when the Metaphit‐treated tissue is subjected to repeated washings before determination of the binding of [3H]threo‐ (±)‐methylphenidate. This finding suggests that Metaphit may be a useful tool in the study of the molecular basis of stimulant action.
Tyrosine Hydroxylase Activity in Caudate Nucleus from Parkinson's Disease: Effects of Iron and Phosphorylating Agents
Authors: Rausch Wolf‐Dieter , Hirata Yoko , Nagatsu Toshiharu , Riederer Peter , Jellinger Kurt
Published: December 2006

Abstract: Tyrosine hydroxylase (TH) activity of human postmortem brain tissues from controls and patients with Parkinson's disease (PD) was examined in the presence of Fe2+ and phosphorylation agents, such as cyclic AMP, exogenous protein kinase, calcium plus calmodulin (Ca2+‐CaM), and ATP. TH activity from parkinsonian tissue was increased by 48% with statistical significance in the presence of exogenous protein kinase. Cyclic AMP alone had no effect, whereas Ca2+‐CaM increased the activity by only 10%. The presence of acetylcholine resulted in a slight decrease in enzyme activity. Human TH was stimulated 13.17‐fold in the presence of 1 mM Fe2+. For iron dependence, no significant differences could be shown for the Km values of TH in striata of PD, while the activity of TH was half of that of controls. Here stimulation with 1 mM Fe2+ raised the activity of TH 11‐fold. Stimulation of rat, gerbil, pig, and human caudate nucleus TH with Fe2+ shows remarkable species differences. In particular, the sensitivity of human TH to stimulating processes is noteworthy. H2O2 decreases TH activity only at high concentrations. Species differences are noted for the combined incubation of Fe2+ and H2O2. In the gerbil caudate nucleus, H2O2 does not prevent the stimulating properties of Fe2+, while the pig shows a dose‐dependent decline of TH activity. In conclusion, there are no significant changes in the stimulating properties of human caudate nucleus TH activity with Fe2+ in PD, while such differences are noted by using exogenous protein kinase. Furthermore, experimental evidence shows that TH activity declines at high concentration of H2O2 only. Potentiation of this effect by Fe2+ seems to be species‐dependent.
Degradation of Thyrotropin‐Releasing Hormone and Luteinising Hormone‐Releasing Hormone by Enzymes of Brain Tissue
Authors: O'Cuinn Gerard , O'Connor Brendan , Elmore Moira
Published: December 2006

Abstract: In this article, the enzymes of brain and associated tissues that can degrade thyrotropin‐releasing hormone (TRH) and luteinising hormone‐releasing hormone (LH‐RH) are reviewed. As both TRH and LH‐RH are considered to act as neurotransmitters or neuromodulators in the CNS, attention is paid to the subcellular location of the enzymes described and how their topographies and substrate specificities fit them to playing roles as inactivating agents for TRH and LH‐RH or as regulators of intracellular concentrations of TRH and LH‐RH. Consideration is also given to enzymes involved in biotransformation of TRH to secondary metabolites that exhibit biological activity and to enzymes involved in the metabolism of secondary metabolites.
Regulation of Ca2+/Calmodulin‐Dependent Protein Kinase II by Brain Gangliosides
Authors: Fukunaga Koji , Miyamoto Eishichi , Soderling Thomas R.
Published: December 2006

Abstract: Purified rat brain Ca2+/calmodulin‐dependent protein kinase II (CaM‐kinase II) is stimulated by brain gangliosides to a level of about 30% the activity obtained in the presence of Ca2+/calmodulin (CaM). Of the various gangliosides tested, GT1b was the most potent, giving half‐maximal activation at 25 μM. Gangliosides GD1a and GM1 also gave activation, but asialo‐GM1 was without effect. Activation was rapid and did not require calcium. The same gangliosides also stimulated the autophosphorylation of CaM‐kinase II on serine residues, but did not produce the Ca2+‐independent form of the kinase. Ganglioside stimulation of CaM‐kinase II was also present in rat brain synaptic membrane fractions. Higher concentrations (125‐250 μM) of GT1b, GD1a, and GM1 also inhibited CaM‐kinase II activity. This inhibition appears to be substrate‐directed, as the extent of inhibition is very dependent on the substrate used. The molecular mechanism of the stimulatory effect of gangliosides was further investigated using a synthetic peptide (CaMK 281‐309), which contains the CaM‐binding, inhibitory, and autophosphorylation domains of CaM‐kinase II. Using purified brain CaM‐kinase II in which these regulatory domains were removed by limited proteolysis, CaMK 281‐309 strongly inhibited kinase activity (IC50=0.2 μM). GT1b completely reversed this inhibition, but did not stimulate phosphorylation of the peptide on threonine‐286. These results demonstrate that GT1b can partially mimic the effects of Ca2+/CaM on native CaM‐kinase II and on peptide CaMK 281‐309.
Solubilization and Partial Purification of α‐Amino‐3‐ Hydroxy‐5‐Methyl‐4‐Isoxazolepropionic Acid Binding Sites from Rat Brain
Authors: Hunter C. , Wheaton K. D. , Wenthold R. J.
Published: December 2006

Abstract: α‐Amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid (AMPA) binding sites were solubilized from rat brain membranes using 1% Triton X‐100 in 0.5 M potassium phosphate buffer containing 20% glycerol. The solubilized binding sites were stable, permitting biochemical and pharmacological characterization as well as partial purification. Pharmacological and binding analyses indicated that the solubilized binding sites were similar to the membrane‐bound sites. Both the solubilized and the membrane‐bound preparations contained high‐ and low‐affinity AMPA binding sites in the presence of potassium thiocyanate. A similar rank order for inhibition of [3H]AMPA binding by several excitatory amino acid analogs was obtained for the soluble and membrane‐bound preparations. [3H]AMPA binding to both soluble and membrane‐bound preparations was increased in the presence of potassium thiocyanate. The solubilized AMPA binding sites migrated as a single peak with gel filtration chromatography, with an Mr of 425,000. Beginning with the solubilized preparation, AMPA binding sites were purified 54‐fold with ion‐exchange chromatography and gel filtration. The characterization and purification of these soluble binding sites is potentially useful for the molecular characterization of this putative excitatory amino acid receptor subtype.
Phorbol Esters Enhance Exocytosis from Chromaffin Cells by Two Mechanisms
Authors: Bittner Mary A. , Holz Ronald W.
Published: December 2006

Abstract: Treatment with phorbol esters such as 12‐O‐tet‐radecanoylphorbol acetate (TPA) rapidly enhances [3H]norepinephrine secretion from digitonin‐permeabilized adrenal chromaffin cells. When TPA treatment was prolonged for several hours, a second distinct enhancing effect was observed. This later enhancement was most prominent at intracellular Ca2+ concentrations of 3–30 μM, and did not require the continued presence of membrane‐bound protein kinase C for its expression. The effect could be elicited by as little as 30‐min exposure to TPA, followed by several hours in TPA‐free medium. This effect of TPA was blocked by actinomycin D and cycloheximide, indicating a requirement for RNA and protein synthesis. Similar effects were seen when intact cells that had been pretreated with TPA were stimulated to secrete by depolarizing concentrations of K+. Thus, protein kinase C enhances secretion by two mechanisms. One is rapid and probably reflects the effects of immediate protein phosphorylation. The other occurs over several hours and requires gene transcription and protein synthesis.
Angiotensin II Effects on the Cytosolic Free Ca2+ Concentration in N1E‐115 Neuroblastoma Cells: Kinetic Properties of the Ca2+ Transient Measured in Single Fura‐2‐Loaded Cells
Authors: Monck Jonathan R. , Williamson Robert E. , Rogulja Ivo , Fluharty Steven J. , Williamson John R.
Published: December 2006

Abstract: The effect of angiotensin II on the cytosolic free Ca2+ concentration was measured in single mouse neuroblastoma N1E‐115 cells loaded with fura‐2. Angiotensin II induced a transient concentration‐dependent increase in Ca2+ and also increased the production of inositol polyphosphates. The Ca2+ increase did not require extracellular Ca2+ and was unaffected by pretreatment with pertussis toxin. These data suggest that angiotensin II increased Ca2+ by an inositol tris‐phosphate‐mediated release of intracellular Ca2+ following activation of phospholipase C via a pertussis toxin‐insensitive guanine nucleotide binding protein. Similar results were obtained with bradykinin. The angiotensin II‐ or bradykinin‐induced increase in Ca2+ occurred after a concentration‐dependent latent period. Low concentrations of agonist elicited a small increase in Ca2+ following a variable lag that sometimes exceeded 1 min, whereas at maximally effective angiotensin II concentrations a larger, more rapid increase in Ca2+ occurred without a measurable delay. In some cells, oscillatory increases in Ca2+ were induced by angiotensin II and bradykinin. Possible mechanisms to explain the concentration dependency of the latent period and the oscillatory nature of the increases of Ca2+ are discussed. These results indicate that the mouse neuroblastoma N1E‐115 cell represents a useful model for studying the signal response transduction mechanisms regulating the effects of angiotensin II in neuronal cells.
Short‐Term Inhibitory Effect of Estradiol on Tyrosine Hydroxylase Activity in Tuberoinfundibular Dopaminergic Neurons In Vitro
Authors: Pasqualini Catherine , Guibert Bernard , Leviel Vincent
Published: December 2006

Abstract: The short‐term inhibition by estradiol of tyrosine hydroxylase (TH) in tuberoinfundibular dopaminergic neurons was examined in vitro on hypothalamic slices from ovariectomized rats. TH activity (determined by L‐3,4‐di‐hydroxyphenylalanine accumulation in the median eminence after blockade of decarboxylase with NSD 1055) showed a 30–40% decrease within 1 h of incubation with estradiol. To determine whether a dephosphorylation process was involved in this decline in TH activity, we studied the sensitivity of the enzyme to dopamine (DA) feedback inhibition: In controls, we observed that two kinetically different forms of TH coexisted, with one exhibiting a Kl(DA) of 26.4 ± 2 μM the other being ∼ 10‐fold more sensitive to DA inhibition, with a [k1{DA)] of 2.56 ± 0.17 μM. likely corresponding to a phosphorylated and active form and to a non‐phosphorylated and poorly active form, respectively. Conversely. after estradiol treatment all TH molecules exhibited the same K1(DA) of 2.5 ± 0.3 μM. This effect was stereospecific, because 17α‐estradiol could not promote it. whereas with 17β‐estradiol. it could be observed at only 10−11M and after a short delay (30 min). Finally, this decrease in the K1(DA) of the purported active form of TH could be prevented by okadaic acid (an inhibitor of protein phosphatases). These results suggest that estradiol can act directly on the mediobasal hypothalamus to trigger a rapid decline in TH activity and that this action may involve a decrease in TH phosphorylation.
Developmental Changes in Expression of a 60‐kDa Somatostatin Receptor Immunoreactivity in the Rat Brain
Authors: Theveniau Magali , Reisine Terry
Published: December 2006

Abstract: The neuropeptide somatostatin (SRIF) exerts several important physiological actions in the adult CNS through interactions with membrane‐bound receptors. SRIF expression is developmentally regulated and this regulation is most apparent in the cerebellum, where SRIF immunoreactivity is expressed at early postnatal ages and then disappears toward adulthood. The transitory nature of SRIF expression at a time of major changes in cerebellum suggests that this peptide may have a role in cerebellar development. To further investigate the role of the SRIF transmitter system during development, we have examined the levels of expression of SRIF receptors in the developing rat brain by immunoblotting using antiserum selective for a 60‐kDa brain SRIF receptor. In whole rat brain, SRIF receptor immunoreactivity first appears at embryonic day 13 (E13), is elevated at E16. increases at birth, peaks at early postnatal ages, and then gradually declines with age. No apparent changes in size of the receptor occur with age. No consistent changes in levels of SRIF receptor immunoreactivity are detected from early postnatal ages to adulthood in the hippocampus, cerebral cortex, and striatum, but levels gradually decline in the hypothalamus. In contrast, SRIF receptor immunoreactivity is expressed transiently in cerebellum. SRIF receptor immunoreactivity is detectable in cerebellum at E16, increases in levels at birth, is apparent from postnatal day 3 to postnatal day 8, and then disappears. The transitory nature of SRIF receptor expression in cerebellum is unique and parallels the expression of SRIF immunoreactivity in this brain region. These findings support the hypothesis that SRIF has a role in cerebellar development.
Vascular α1‐adrenoceptors in isolated perfused rat kidney: influence of ageing
Authors: Awe S. O. , Adeagbo A. S. O.
Published: December 2006

1 The present study identifies α1‐adrenoceptor subtype(s) involved in constrictor responses of the kidney and how ageing influences it.

2 The study was conducted on kidneys from F344BNF1 rats, which unlike F344 or Wistar rats used by many previous investigators do not exhibit glomerulonephritis at advanced age.

3 Noradrenaline (NA) and phenylephrine (PHE) (non‐selective α1) and A61063 (selective α1A) adrenoceptor agonists elicited constriction of perfused kidneys of young and old rats. The pD2 values (index of renovascular reactivity) were significantly higher for A61603 than for either PHE or NA, and significantly decrease across age groups.

4 BMY 7378 or RS 100329, α1D‐ or α1A‐adrenoceptor antagonists, respectively antagonized the constrictor responses and suppressed the maximal responses to all agonists in young adult rat kidneys. However, antagonism of PHE or A61063 by BMY 7378 in old rat kidneys was surmountable.

5 This study suggests that: (i) α1A and α1D‐adrenoceptor subtypes mediate vasoconstriction of perfused rat kidney; (ii) α1A‐adrenoceptor subtype appears to predominate in renal vasculature based on agonist relative potencies. (iii) Ageing significantly decreases α1‐adrenoceptor‐mediated vasoconstriction of rat kidney.
The classical competitive antagonism of phentolamine on smooth muscle preparations, investigated by two procedures
Authors: Patil P. N.
Published: December 2006

1 In isolated smooth muscle tissues taken from rats, rabbits and guinea‐pigs, all at 37.5 °C, the equilibrium dissociation constant (K) of the competitive, reversible α‐adrenoceptor antagonist phentolamine varied between 4 and 28 nm.

2 The concentration of the antagonist required to inhibit contractions to direct‐ or indirect‐acting α‐adrenenoceptor agonists by 50% (IC50) also varied between 5 and 30 nm.

3 From one tissue to another, the IC50/K ratio of the blocker varied from 1 to 2.5, the values being close to those predicted by classical receptor theory based on the law of mass action.

4 At 27.5 °C, using phenylephrine as the spasmogen in rat aorta, the IC50/K ratio for phentolamine was 3.1.

5 A significantly higher IC50 compared with K for phentolamine indicates that the procedures for estimating affinity constants for a competitive antagonist are not equivalent.
Heterogeneous effect of leucotriene CysLT1receptor antagonists on antigen‐induced motor and inflammatory responses in guinea‐pig airways
Authors: Alfieri A. B. , Tramontana M. , Cialdai C. , Lecci A. , Giuliani S. , Crea A. , Manzini S. , Maggi C. A.
Published: December 2006

1 The effect of montelukast or MEN91507, selective leucotriene CysLT1receptor antagonists, on antigen‐induced airway inflammation and bronchoconstriction were compared in anaesthetized guinea‐pigs.

2 In sensitized animals, ovalbumin (0.3 mg kg−1, i.v.)‐induced microvascular leakage in trachea, intrapulmonary airways, total lung (parenchyma and intrapulmonary airways) and urinary bladder was reduced by MEN91507 (0.01–1 μmol kg−1, i.v.), whereas montelukast (0.01–1 μmol kg−1, i.v.) antagonized the effect of the antigen only in the lung and urinary bladder.

3 Ovalbumin (1 mg kg−1, i.v.)‐induced bronchoconstriction was dose dependently antagonized by MEN91507 (10–30 μmol kg−1, i.v.), whereas the effect of montelukast (0.1–30 μmol kg−1, i.v.) was marginal (15–30% inhibition). Neither MEN91507 nor montelukast (30 μmol kg−1, i.v.) affected the bronchoconstrictor response induced by acetylcholine (0.3 μmol kg−1, i.v.) in sensitized animals.

4 It is concluded that montelukast and MEN91507 display a differential activity against the effect of endogenous leucotrienes, despite the fact that both compounds show a similar antagonist profile against exogenous leucotrienes acting through CysLT1receptors.
In vivoeffects of amtolmetin guacyl on lipid peroxidation and antioxidant defence systems in different models of gastrointestinal injury
Authors: Kirkova M. , Kesiova M. , Konstantinova S. , Alexandrova A. , Petrov L. , Tsvetanova E. , Todorov S.
Published: December 2006

1 Thein vivoeffects of the non‐steroid anti‐inflammatory drug (NSAID) amtolmetin guacyl (AMG) on lipid peroxidation (LP) and on antioxidant enzyme and non‐enzyme defence systems were investigated in models of stomach and colon damages, induced by other NSAIDs, by ethanol or by 2,4,6‐trinitrobenzenesulfonic acid (TNBS).

2 Indomethacin increased LP, glutathione peroxidase (GSH‐PX) and glucose‐6‐phosphate dehydrogenase (Glu‐6‐P‐DH) activities and decreased glutathione levels in gastric mucosa. Pretreatment with AMG normalized some of the parameters affected by indomethacin.

3 Treatment of rats with ethanol for 0.5 h led to a decrease in glutathione levels as well as activities of glutathione reductase and Glu‐6‐P‐DH in gastric mucosa. AMG, administered 0.5 h before ethanol, limited the adverse actions of ethanol.

4 Amtolmetin guacyl failed to abolish the TNBS‐induced changes in the followed‐up parameters in colon mucosa and liver, but additional alterations (as with tolmetin) were not observed.

5 The beneficial profile of AMG in the various experimental models of free radical‐induced damage investigated in this study suggests the possibility that this drug might possess antioxidant activity.
Late Results of Gelatin–Resorcin–Formalin Glue‐aided Repair in Acute Type A Aortic Dissection
Authors: Shiono Motomi , Hata Mitsumasa , Sezai Akira , Niino Tetsuya , Yagi Shinya , Negishi Nanao
Published: December 2006

Abstract:  Gelatin–resorcin–formalin (GRF) glue has been used to obliterate the false lumen of dissected aortas, resulting in reduced mortality. However, because of the cytotoxicity of formalin, the application of GRF remains controversial. In this study, a total of 138 consecutive patients with acute type A dissection since 1995, who underwent emergency graft replacement, were reviewed. The mean age was 65.5 years. The hospital mortality rate was 6.5%. In‐hospital re‐exploration rate and patency rate of the false lumen were 6.5% and 24.7%, respectively. The actuarial survival rates were 81.5% after 5 years and 54.8% after 10 years. Reoperation‐free rates were 87.9% after 5 years and 72.3% after 10 years. Tissue necrosis or aneurismal degeneration was not demonstrated at reoperation. In conclusion, GRF glue demonstrats excellent tissue adhesion and hemostasis capability, and contributes to improve surgical results.
Can Hip Protector Use in the Nursing Home Be Predicted?
Authors: Honkanen Lisa A. , Monaghan Niall , Reid M. C. , Newstein David , Pillemer Karl , Lachs Mark S.
Published: December 2006

OBJECTIVES: To identify resident‐ and caregiver‐related factors that influence hip protector (HP) usage.

DESIGN: Prospective observational cohort study over 6 weeks.

SETTING: A 409‐bed nursing home located in New York City.

PARTICIPANTS: Fifty‐two residents and 60 certified nursing assistants (CNAs).

INTERVENTION: Free HPs were provided to residents enrolled in the study.

MEASUREMENTS: HP adherence (number of hours HPs were worn/shift); HP users (subjects who used HPs for more than 1 hour during any 8‐hour shift as recorded by CNAs in week 6 or who used HPs at checks performed by research staff in week 6 and intended future use); CNAs' scores on a questionnaire quantifying osteoporosis knowledge, opinions on HPs, job satisfaction, and health locus of control.

RESULTS: Sixty‐eight percent of residents were HP users. Factors independently associated with this outcome included male sex, age, Parkinson's disease, family involvement, impaired cognition, dependent ambulation, and CNAs' sense that chance affects health outcomes. In multivariate models, anxiety, resistive behaviors, ambulation, and fall history in residents and CNAs' perceptions of HPs and their sense of how chance or other staff determine health outcomes predicted HP adherence.

CONCLUSION: Resident‐related factors and CNAs' sense of external controls predicted HP users and HP adherence. CNAs' understanding of osteoporosis and HP affected only HP adherence. Most resident‐related factors were not remediable but might help to target likely HP users. To improve HP adherence, educational programs should be designed to improve CNAs' understanding of osteoporosis and HP. Larger studies are needed to confirm these findings.