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1.
The incorporation of radioactive label into the myelin basic protein isolated from whole brain and from purified myelin of Quaking mice and normal littermates was compared. Four Quaking mice (32 days) and 4 littermate controls were injected intracranially with 150 μCi [2- 2H]glycine and 25 μCi of [2- 14C]glycine, respectively. One hour later, the 8 mice were sacrificed and their brains combined for common homogenization. The 3H/ 14C ratios of the small and large basic proteins in whole brain were 3.44 and 2.48 respectively, while the 3H/ 14C ratios for these proteins in myelin were 0.79 and 1.00, respectively. In the same experiment, the microsomal fraction had a 3H/ 14C ratio of 2.98 which is the expected ratio for normal incorporation. The results indicate that the synthesis of basic protein in whole brain of Quaking mouse proceeds at a normal rate, but specifically, the incorporation of basic protein into myelin is depressed suggesting a defect at the step of assembly of myelin components into a final membrane product. 相似文献
2.
The binding of [ 3H]nipecotic acid to frozen post-mortem human brain tissue has been characterized. Competition experiments with γ-aminobutyric acid (GABA), GABA uptake inhibitors, ligands active at post-synaptic GABA receptors and receptors for other neurotransmitter systems, suggest that [ 3H]nipecotic acid binds to the neuronal (but not glial) GABA uptake site. Competition and kinetic experiments suggest that 85% of the binding is to high affinity site. The dissaciation constants (Kd) measured in kinetic and equilibrium experiments were in the same range (0.5–0.6 μM). The regional distribution was studied in 19 brain regions and the binding was relatively homogenous. It is concluded that [ 3H]nipecotic acid binding can be used as a marker for neuronal GABA uptake sites in post-mortem human brain tissue. 相似文献
3.
To address the question whether the changes in cortical glucose metabolism observedin patients with Alzheimers disease are interrelated with, or consequences of, basal forebraincholinergic cell loss, an experimental approach was employed to produce cortical cholinergicdysfunction in rat brain by administration of the cholinergic immunotoxin 192IgG-saporin. [ 14C]
-glucose utilization in brain homogenates,
-glucose-displaceable [ 3H]cytochalasin B binding to glucose transporters (GLUT),Northern and Western analyses, as well as in vivo [ 14]2-deoxyglucoseautoradiography were used to quantify the regional glucose metabolism. Basal forebrain cholinergic lesion resulted in transient increases in glucose transporterbinding in cortical regions displaying reduced acetylcholinesterase activity, already detectableseven days after lesion with peak values around 30 days post lesion. Western analysis revealedthat the changes in total glucose transporter binding are mainly due to changes in the GLUT3subtype only, while the levels of GLUT1 and GLUT3 mRNA (Northern analysis) were notaffected by cholinergic lesion. Both immunocytochemistry and in situ hybridizationdemonstrated preferential localizations of GLUT1 on brain capillaries and GLUT3 on neurons,respectively. A lesion-induced transient decrease in [14C]
-glucoseutilization seven days post lesion was detected in the lesion site, whereas cholinoceptive corticalregions were not affected. In vivo [14C]deoxyglucose uptake was transientlyincreased in cholinoceptive cortical regions and in the lesion site being highest between three toseven days after lesion. The cholinergic lesion-induced transient up-regulation of cortical glucose transporters anddeoxyglucose uptake reflects an increased glucose demand in regions depleted by acetylcholinesuggesting functional links between cortical cholinergic activity and glucose metabolism incholinoceptive target regions. 相似文献
4.
The defect in myelinogenesis present in the Quaking mutant mouse was investigated using a double radioisotope technique for comparing the incorporation of amino acid into myelin proteins of normal and mutant mice. Quaking mice and littermate controls recieved intracranial injections of 150 μCi [ 3H]glycine and 25 μCi of [ 14C]glycine respectively. After 2 h their brains were combined and jointly processed to obtain subcellular fractions. The 3H/ 14C ratio for the myelin subfraction was 1.88 as compared to a 3H/ 14C ratio of 3.0 for the other subfractions, indicating a 40% decrease in glycine incorporation into myelin of Quaking mice. Myelin proteins were separated by discontinuous gel electrophoresis in the presence of sodium dodecyl sulfate (SDS) and the 3H/ 14C ratios determined in each gel slice. In contrast to the microsomal subfractions which gave a 3H/ 14C ratio of 2.6 across the gel, the 3H/ 14C ratio of myelin showed large variations with values ranging from 0.54 for proteolipid protein to 2.0 for some of the high molecular weight proteins. During development, the Quaking mutant exhibited a preferential depression in glycine incorporation into proteolipid protein in 18-day-old mice, while in older animals (32–54 days) the fast migrating basic protein, as well as the proteolipid protein, was labeled to a significantly lesser extent. 相似文献
5.
Although indirect pharmacologic evidence has suggested the presence of a lipoxygenase pathway of arachidonic acid (AA) metabolism in blood vessels, direct biochemical evidence has been difficult to demonstrate. We have investigated lipoxygenase metabolism in both fresh vessel preparations and cultured vascular cells from various sources and species. Lipoxygenase-derived [ 3H]HETE (composed of 12-HETE, 15-HETE and 5-HETE), which was abolished by ETYA but not by aspirin, was formed when [ 3H]AA was incubated with fresh sections of rat aorta. Lipoxygenase activity was lost following deendothelialization. A single peak of [ 3H]15-HETE was produced by cultured bovine aortic and human umbilical vein endothelial cells (EC) in response to exogenous [ 3H]AA or from [ 3H]AA released by ionophore A23187 from endogenous EC membrane phospholipid pools. Cultured bovine, rabbit or rat aorta smooth muscle cells had no detectable 15-lipoxygenase activity. 14C]Linoleic acid was converted by EC to its 15-lipoxygenase metabolite, [ 14C]13-hydroxyoctadecadienoic acid. These results indicate that blood vessels from different sources and species have a 15-lipoxygenase system, and this activity resides predominantly in the endothelial cells. 相似文献
6.
A sensitive chromatographic procedure was used to study the metabolism of [ 14C]tyrosine, [ 3H]DOPA and [ 3H]dopamine in 3 defined cell-types situated in the nervous system of Planorbis corneus. One of the cell-types contains dopamine (GDC), the other serotonin (GSC) and the other neither amine (GC). The GDCs metabolise [ 14C]tyrosine to form DOPA and dopamine while the other two cells lack this ability. In contrast, the GDCs and the GSC, but not the GCs, metabolise [ 3H]DOPA to form dopamine. In addition the GDCs incorporate radioactivity from [ 3H]DOPA into DOPAC, homovanillic acid and methoxytyramine. After incubation of cells in [ 3H]dopamine, only the GDCs metabolise it to form DOPAC, homovanillic acid and methoxytyramine. In no instance did the GDCs form significant amounts of noradrenaline from the incorporated radioactive substances. These results, together with data on the amine histochemistry of the individual cell-types following pretreatment of animals with drugs known to affect specific enzymes in the synthesis of amine transmitter substances, clearly demonstrate that the GDCs alone have the enzymes requisite for the biosynthesis and catabolism of dopamine, but not noradrenaline. 相似文献
7.
Astrocyte-enriched primary cultures of newborn rat brain hemispheres, prelabeled with [ 3H]inositol, accumulated [ 3H]inositol phosphate but not [ 3H]inositol bis-and tris-phosphate, after exposure to histamine for 60 min in the presence of 10 mM LiCl. The response to histamine was not a function of contaminating meningeal fibroblasts since no accumulation of [ 3H]inositol phosphate was elicited by histamine in meningeal cultures. The stimulation of phosphoinositide hydrolysis by histamine in astrocytes was dose-dependent (EC 50 = 1.7 μM, maximal effect = 345% over basal levels) and was mimicked by several H 1-receptor agonists. The use of selectiver receptor antagonists confirmed that the histamine response was the result of activation of H 1-receptors. The histamine-induced [ 3H]inositol phosphate accumulation was completely abolished by omission of Ca 2+ from the incubation medium. Astrocyte membranes specifically bound the radiolabeled H 1-antagonist, [ 3H]mepyramine with an affinity ( Kd = 5.9 nM) and a density of binding sites ( Bmax = 113 fmol/mg protein) similar to rat brain. These results demonstrate the presence of functional histamine H 1-receptors in rat brain astrocytes and suggest a role for histamine as a neuromodulator of astrocyte function. 相似文献
8.
The effects of phosphatidylserine (PS) on the binding properties of the AMPA (-amino-3-hydroxy-5-methylisoxazolepropionic acid) and NMDA ( N-methyl-d-aspartate) subtypes of glutamate receptors were analyzed by quantitative autoradiography of [ 3H]AMPA, [ 3H]6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and [ 3H]glutamate binding on at brain tissue sections. Preincubation of brain sections with PS produced an increase in [ 3H]AMPA binding without modifying the binding properties of [3H]CNQX, an antagonist of AMPA receptors. This effect of PS appeared to be specific for the AMPA subtype of glutamate receptors as the same treatment did not modify [ 3H]glutamate binding to the NMDA receptors. Furthermore, the PS-induced increase in [3H]AMPA binding was different in various brain structures, being larger in the molecular layer of the cerebellum and almost absent in the striatum. Preincubation with calcium also augmented [ 3H]AMPA binding, and the lack of additivity of the effects of calcium and PS on [ 3H]AMPA binding strongly suggests that both treatments share a common mechanism(s) for producing increased agonist binding. Finally, the effect of PS on AMPA receptor properties was markedly reduced in rat brain sections prepared from neonatal rats at a developmental stage that is normally characterized by the absence of LTP expression in certain brain regions. The present data are consistent with the hypothesis that alteration in the lipid composition of synaptic membranes may be an important mechanism for regulating AMPA receptor properties. which could be involved in producing long-lasting changes in synaptic operation. 相似文献
9.
Mouse brain membrane vesicles (microsacs) were transiently permeabilized by hypo-osmotic shock. This permeabilization method resulted in the encapsulation of both [ 14C]sucrose and exogenous alkaline phosphatase. The efficiency of this method was estimated by [ 14C]sucrose encapsulation experiments to be approximately 81%. External membrane binding experiments with the lectin [ 3H]concanavalin A demonstrate that the microsacs were not inverted by permeabilization. Following permeabilization, the functional integrity of a ligand-gated ion channel, the GABA A receptor complex, was investigated. Muscimol-stimulated 36Cl − uptake experiments show that this receptor retains its functional properties including blockade by the receptor antagonist bicuculline and potentiation by the allosteric modulators flunitrazepam and pentobarbital. The osmotic permeabilization technique described here provides several advantages over other permeabilization methods. These advantages include a high trapping efficiency, the encapsulation of not only small solutes but large membrane impermeant compounds such as enzymes and the functional preservation of at least one transmembrane protein. Furthermore, this method does not require specialized equipment and does not result in large, permanent holes in the plasma membrane. 相似文献
10.
The ability of astrocytes to sequester MeHG may indicate an astrocyte-mediated role in MeHg's neurotoxicity. Hence, studies were undertaken to assess the effects of MeHg on metabolic functions in cultured astrocytes. MeHg (10 −5 M) significantly inhibited the initial rate (5 min) of uptake of 86RbCl, used as a tracer for K +. 86RbCl uptake was also sensitive to the omission of medium Na +. MeHg (10 −5 M) also markedly inhibited the initial rate of uptake (1 min) of the Na +-dependent uptake of [ 3H]l-glutamate. A second neurotoxin, MnCl 2 (0–5 × 10 −4 M), did not alter [ 3H]glutamate or 86RbCl uptake. MeHg, but not MnCl 2, also stimulated the release of intracellular 86Rb + in a dose-dependent fashion. This effect could be prevented by the administration of MeHg as the glutathione conjugate. These observations support the hypothesis that the astrocyte plasma membrane is an important target for MeHg's toxic effect and specifically that small concentrations of this organometal inhibit the ability of astrocytes to maintain a transmembrane K + gradient. This would be expected to compromise the ability of astrocytes to control extracellular K + either by spatial buffering or active uptake, resulting in cellular swelling. We therefore studied volume changes in astrocytes using uptake of [ 14C]3-O-methyl-d-glucose, in attached cells in response to exposure to MeHg. Exposure to MeHg (0–5 × 10 −4 M) caused a marked increase in the cell volume that was proportional to concentrations of MeHg. 相似文献
11.
1. We have characterized and quantified specific binding of [ 3H]-flunitrazepam (FNZ: (benzodiazepine), [ 3H]-naloxone (NAL: (opiate) and [ 3h]cgp-12177(CGP: (beta-adrenergic) to thick slices (230–400 μm) of mouse and rat brain. 2. The binding sites are stereospecific, saturable and of high affinity. In all cases, the binding of the ligands is readily reversible and demonstrates the appropriate drug specificity. 3. In mouse brain [3H]-NAL binding is elevated by chronic treatment with naloxone (via capsules). 4. We have been unsuccessful in quantifying beta adrenoreceptors with the archetypal ligand [3H]-dihydroalprenolol (DHA). However, the use of [3H]-CGP 12177 enabled us to detect high-affinity beta adrenoreceptors in brain slices. 5. [3H]-CGP also permits the demonstration of rapid and reversible agonist-induced down-regulation (internalization) of beta binding sites. 6. We have been successful in quantifying beta adrenergic sites in single pineal glands of rat and hamster. 相似文献
12.
Selective retrograde labelling with [ 3H]serotonin ([ 3H]5-HT) can be used to identify serotonergic cell bodies after specific [ 3H]5-HT uptake by the corresponding nerve terminals. In the present study, we demonstrate that autoradiography of this [ 3H]5-HT radiolabelling can be combined with immunocytochemical detection of endogenous serotonin, GABA or substance P on the same tissue section. The midbrain raphe serotonergic projections to the olfactory bulb and the spinal projections of medullary serotonergic nuclei were investigated. The specificity of retrograde labelling with [ 3H]5-HT was confirmed by immunoreactivity of the radiolabelled cells for serotonin, using an antiserum specific for formaldehyde-fixed serotonin. After spinal injections of [ 3H]5-HT, many retrogradely labelled cells in the medullary raphe were immunopositive for substance P, and a few for GABA. These results are in agreement with the available information on the co-existence of putative transmitters in the spinal projections of caudal raphe neurons. Therefore, autoradiography of [ 3H]5-HT retrograde labelling combined with immunocytochemistry offers a possibility to test the specificity of transmitter-selective retrograde labelling, to identify transmitter-defined neuronal interactions and to investigate the projection fields of multitransmitter containing neurons. 相似文献
13.
Serotonin (5-hydroxytryptamine; 5-HT), acetylcholine and γ-aminobutyric acid (GABA) are neurotransmitters in the rat visual system. Using quantitative autoradiography, the effect of unilateral orbitral enucleationon [ 3H]5-HT, [ 3H]ketanserin, [ 3H]quinuclidinyl benilate (QNB) and [ 3H]muscimol binding to 5-HT 1, 5-HT 2, muscarinic and GABA A receptors has been examined within anatomical components of the visual pathway at 4 time points up to 20 days after the lesion. The functional deficit was assessed in the same animals using quantitative [ 14C]2-deoxyglucose autoradiography. At 1 day after unilateral orbital enucleation, there were no significant alterations in ligand binding although local cerebral glucose use was reduced in primary visual structures in the visually deprived hemisphere. At 5 days post-enucleation, however, [ 3H]5-HT binding was significantly reduced in both the visually deprived superior colliculus (by 17%) and dorsal lateral geniculate body (DLG) (by 33%). There were similar alterations in the binding of this ligand in these primary retinal projections areas at 10 and 20 days after orbital enucleation, but there were no changes in secondary areas (e.g. visual cortex) at any time point. [ 3H]Muscimol binding was significantly reduced in the visually deprived DLG (30%) and visual cortex (21%) only at 20 days post-lesion, whilst [ 3H]ketanserin and [ 3H]QNB were not altered in any region in the visually deprived hemisphere at any time point post-enucleation. At 10 and 20 days post-enucleation, the degree of [ 3H]5-HT, and [ 3H]muscimol binding deficits in visually deprived structures correlated significantly with the level of reduced metabolic activity in these areas ( r = 0.700andr = 0.543respectively). The specificity and regional and temporal heterogeneity of neurotransmitter receptor binding alterations provides evidence of selective adjustments within visual system component6s in response to orbital enucleation. 相似文献
14.
Effects of protein kinase C (PKC) activation on the function of the GABA/benzodiazepine receptor-chloride complex were analyzed by quantitative autoradiography using [ 3H]muscimol, [ 3H]flunitrazepam and [ 35S]TBPS in rat brain slices. The density of [ 3H]muscimol binding was highest in cerebellar granular layers and high in both the frontal cortex and thalamus, but binding levels in the hippocampus were low. After activation of PKC by 100 nM phorbol-12,13-dibutyrate (PDBu), [ 3H]muscimol binding was decreased in the frontal cortex, striatum and thalamus, but binding levels were not changed in the hippocampus or cerebellum. The density of [ 3H]flunitrazepam binding was high in the cortex, hippocampus and molecular layers of cerebellum but was low in thalamus. PDBu increased the [ 3H]flunitrazepam binding only in the striatum and in part of the cortex and thalamus after activation of PKC. After activation of PKC by PDBu, [ 35S]TBPS binding was increased in most areas, but binding levels were not changed in the brainstem or cerebellum. The receptor binding was markedly decreased in almost all areas by the addition of 2.5 mM Mg 2+. Elevated [ 35S]TBPS binding produced by PDBu was significantly inhibited by the addition of Mg 2+. These results suggest that the activation of PKC potentiates benzodiazepine and TBPS binding, but decreases muscimol binding in a region-specific manner in the rat brain. 相似文献
15.
The benzodiazepine antagonist Ro 15-1788 was labelled with [ 11C] and examined for possible use as ligand for PET scan studies on benzodiazepine receptors in the brain of cynomolgus monkeys and human subjects. [ 11C] Ro 15-1788 allowed the in vivo visualization of benzodiazepine receptor binding in cerebral and cerebellar cortical areas as well as in basal brain nuclei in PET scan images. [ 11C] Ro 15-1788 exhibited a high ratio of specific benzodiazepine receptor binding (cerebral cortex) to non-specific binding (pons) and the kinetics of binding should be satisfactory for quantitative clinical PET scan studies using [ 11C]. The in vivo binding of [ 11C] Ro 15-1788 in the cerebral cortex of cynomolgus monkeys and healthy human subjects was reduced by approximately 90% within 10 min after the intravenous injection of a high dose of unlabelled Ro 15-1788 (0.5 mg/kg i.v.). Different areas of the healthy human brain showed an approximately 10-fold variation in maximal [ 11C] Ro 15-1788 binding that corresponded to the previously known distribution of benzodiazepine receptors in these regions. The highest degree of binding was obtained in the medial occipital cerebral cortex followed by frontal cortex, cerebellum, thalamus, striatum and pons. Two psychiatric patients with anxiety syndromes who had been treated for a long time with high doses of benzodiazepines had roughly the same degree of maximal [ 11C] Ro 15-1788 binding in brain regions as the healthy subjects but the rate of decline of [ 11C] Ro 15-1788 in the brain was higher. This indicates that there is measurable competition between [ 11C] Ro 15-1788 binding and clinical benzodiazepine concentrations in the body fluids of psychiatric patients. The results demonstrate that [ 11C] Ro 15-1788 should be a valuable tool for quantitative analyses of benzodiazepine receptor characteristics and receptor occupancy in the brain of patients with neuropsychiatric disorders. 相似文献
16.
The postnatal development of nicotine-like binding sites in the cortex, hippocampus, midbrain and cerebellum of 3-, 7-, 12-, 17- and 30-day-old mice was studied. Two different nicotinic cholinergic ligands, namely [ 3H]acetylcholine ([ 3H]ACh) and [ 3H]nicotine ([ 3H]NIC) were used to detect the nicotine-like binding sites in in vitro binding assays. The postnatal development of the binding sites of [ 3H]NIC increased gradually with age in all brain regions studied. The [ 3H]ACh binding, on the other hand, showed a marked peak on day 12 in the cerebellum and midbrain but did not change notably with age in the hippocampus and cortex, except for a slight temporary increase in the cortex on day 7. The time-course for the appearance of nicotinic binding sites as observed with [ 3H]ACh was found to be rather similar to that earlier described for [ 3H]alpha-bungarotoxin binding sites, whereas that for [ 3H]NIC differed from that described for other nicotinic ligands. 相似文献
17.
The binding of [ 3H]hemicholinium ([ 3H]HCh-3) to sodium-dependent high-affinity choline uptake sites provides a useful neuroanatomical and functional marker of the cholinergic system. We examined the autoradiographic distribution of [ 3H]HCh-3 binding sites in the forebrain of young (4–6 months) and old (32 months) rats. There was a widespread reduction of [ 3H]HCh-3 binding site density in the aged rat brain. This loss presented regional differences with maximal reduction in the medial and posterior striatum (55%) and in the dentate gyrus (47%), in limbic areas such as basolateral amygdala, tubercle olfactorium and piriform cortex the autoradiographic signal was about 25–30% lower. In aged hippocampus and cerebral cortex the density of [ 3H]HCh-3 binding sites was about 40% lower, the difference between young and senescent animals being less evident in the medial septum and basal nucleus. No significant alterations were observed in interpeduncular nucleus from old rats. These data are in agreement with the functional results obtained by measuring other cholinergic parameters in the aged rat and confirm the vulnerability of cholinergic system during aging 相似文献
18.
Brain abscesses are classical complications of congenital heart disease (CHD) in children and adolescents. This association is rarely observed in adults. We report a 46-year-old man presenting a fronto-parietal abscess associated with an asymptomatic atrial septal defect. Positron emission tomography (PET) study revealed high uptake of l-[ methyl- 11C]methionine ([ 11C]methionine) and 2-[ 18F]fluoro-2-deoxy-d-glucose (FDG) around the brain abscess. We suggest (1) to exclude a silent cardiac malformation in the presence of a cerebral abscess of unknown source occurring in adults; (2) to consider the diagnosis of brain abscess in cases of high uptake of [ 11C]methionine and FDG in relation to a brain lesion. 相似文献
19.
Plasmodium yoelii infected cerebral microvessels of mice had an enhanced time-dependent, temperature-sensitive, and saturable uptake of [ 14C]-amino acid. viz. leucine, valine and glycine. Metabolic inhibitors caused a noticeable inhibition of amino acid uptake in normal microvessels as compared to infected cerebral microvessels indicating that the uptake of [ 14C]-L-leucine, [ 14C]-L-valine and [ 14C]-glycine is an energy dependent process. 相似文献
20.
Three ion channel binding sites were examined by means of quantitative ligand binding autoradiography in temporal and occipital cortex from 9 patients with neuropathologically confirmed Alzheimer's disease (AD) and 7 matched control subjects. The following ligands were used: 125I-apamin to label a population of Ca 2+-sensitive K + channels; [ 3H]PN200-110 to label L-type voltage-sensitive Ca 2+ channels and [ 3H]glibenclamide to label ATP-sensitive K + channels. Ion channel binding sites were compared to: choline acetyltransferase (ChAT) activity and plaque densities measured in the same tissue. In the temporal cortex in AD 125I-apamin binding was increased compared to controls (e.g. superficial layers: control= 0.71 ± 0.07;AD= 1.02 ± 0.07,mean±S.E.M. pmol/g tissue). In contrast, in adjacent sections [ 3H]glibenclamide binding was reduced in AD compared to controls (e.g. superficial layers: control= 25.3 ± 1.7;AD= 17.9 ± 1.4pmol/g tissue). [ 3H]PN200-110 binding in temporal cortex was not altered in AD compared to controls. In the occipital cortex 125I-apamin binding was increased in AD while both [ 3H]glibenclamide and [ 3H]PN-200-110 binding sites in this cortical area were not different from controls. Plaque density (per mm 2) was higher in temporal (e.g. layers I–III, 43 ± 6) than in occipital cortex (layers I–III, 27 ± 4) in the AD patients while ChAT was reduced by 40% in temporal cortex and by 50% in occipital cortex compared to controls. The results suggests that the three ion channel binding sites are located on structural elements in the brain which are differentially affected by the pathophysiology of AD. 相似文献
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