摘要:

Abstract:Polyclonal antibodies were raised to synthetic peptides having amino acid sequences corresponding with the N‐ or C‐terminal part of the γ‐aminobutyric acidA(GABAA) receptor α5‐subunit. These anti‐peptide α5(2–10) or anti‐peptide α5(427–433) antibodies reacted specifically with GABAAreceptors purified from the brains of 5–10‐day‐old rats in an enzyme‐linked immunosorbent assay and were able to dose‐dependently immunoprecipitate up to 6.3 or 13.1% of the GABAAreceptors present in the incubation, respectively. In immunoblots, each of these antibodies reacted with the same two protein bands with apparent molecular mass of 53 or 57 kDa. After exhaustive treatment of purified GABAAreceptors withN‐Glycanase, each of these antibodies identified two proteins with apparent molecular masses of 46 and 48 kDa. Additional treatment of GABAAreceptors with neuraminidase andO‐Glycanase resulted in an apparently single protein with molecular mass of 47 kDa, which again was identified by both the anti‐peptide α5(2–10) and the anti‐peptide α5(427–433) antibody. These results indicate the existence of at least two different α5‐sub‐units of the GABAAreceptor that differ in their carbohydrate content. In contrast to other α‐ or β‐subunits of GABAAreceptors so far investigated, at least one of

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1993.tb05826.x

出版商:Blackwell Publishing Ltd    

年代:1993

数据来源: WILEY

摘要:

Abstract:Three autopsy brains from patients who succumbed to malignant gliomas have been analyzed in various regions with regard to their ganglioside content. The study focused on the gangliosides GD3 and 3′‐isoLM1, which in a previous study of biopsies were found to be associated with these tumors. In particular, 3′‐isoLM1, was suggested to be a marker for malignant gliomas. The highest concentrations (200–1,000 nmol of sialic acid/g wet weight) of GD3 was found in specimens of macroscopically pure tumor, where the proportion of GD3 was, at the most, 78% (range, 11–78%) of the total ganglioside sialic acid compared with10% of the total ganglioside sialic acid (range, 3–37%). The ganglioside 3′‐isoLM1, as determined by TLC‐enzyme‐linked immunosorbent assay using a specific monoclonal antibody (SL‐50), was not present at detectable levels in any of the macroscopically homogenous tumor areas. It was, however, found in the periphery of the tumor, in the corpus callosum, and at highest concentrations in the region of the opposite hemisphere corresponding to the tumor. The concentration varied between 0.1 and 6.0 nmol/g wet weight of tissue. The 3′‐isoLM1 ganglioside was not detected in normal gray or white matter or in the normal corpus callosum, but in one of three breast cancer metastasis, one of two low differentiated cancer metastases, and one stomach cancer. The concentration was 1–4 nmol/g wet weight. These results indicate a unique distribution of the gangliosides GD3 and 3′‐isoLM1 and suggest that they play distinct roles in interact

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1993.tb05827.x

出版商:Blackwell Publishing Ltd    

年代:1993

数据来源: WILEY

摘要:

Abstract:Uptake of59Fe from blood into brains of anaesthetized rats and mice has been studied by intravenous infusion of [59Fe]ferrous ascorbate or of59Fe‐transferrin, the results not being significantly different. Uptakes in the rat were linear with time, but increased at longer times in the mouse. Transfer constants,Kin(in ml/g/h × 103), for cerebral hemispheres were 5.2 in the adult rat and 5.6 in the mouse. TheseKinvalues corresponded to59Fe influxes of 145 and 322 pmol/g/h, respectively.59Fe uptake into the mouse brain occurred in the following order: cerebellum>brainstem>frontal cerebral cortex>parietal cortex>occipital cortex>hippocampus>caudate nucleus. In genetically hypotransferrinaemic mice,59Fe uptake into brain was 80–95 times greater than in To strain mice. Pretreatment of young rats and mice with monoclonal antibodies to transferrin receptors, i.e., the anti‐rat immunoglobulin G OX 26 and the anti‐mouse immunoglobulin M RI7 208, inhibited59Fe uptake into spleen by 94% and 98%, respectively, indicating saturation of receptors. The antibodies reduced59Fe uptake into rat brain by 35–60% and that into mouse brain by 65–85%. Although a major portion of iron transport across the blood‐brain barrier is normally transferrin‐mediated, non‐transferrin‐bound iron readily crosses it at low seru

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1993.tb05828.x

出版商:Blackwell Publishing Ltd    

年代:1993

数据来源: WILEY

摘要:

Abstract:The plasticity of astroglial glutamate and γ‐aminobutyric acid (GABA) uptakes was investigated using mouse cerebellar cell cultures. The influence of external factors, such as different sera and/or the presence of neurons, was examined. Control autoradiography experiments showed that after short‐term exposure to radioactive amino acids, granule cells took up neither glutamate nor GABA, and β‐alanine predominantly inhibited astroglial GABA uptake. Astroglial uptake was quantified by measuring the radioactivity taken up by the cells in the culture and relating this measurement to the number of glial fibrillary acidic protein‐positive cells present. Glutamate uptake was investigated in astroglial cultures and subcultures and in neuro‐nal‐astroglial cultures derived from postnatal day 4 mouse cerebella. In the absence of neurons, glutamate uptake increased during the first 9 days after plating and then leveled off. At 14 days in vitro in horse serum, which favors the differentiation of fibrous‐like astrocytes, glutamate uptake related to astrocyte number was twice as high as in fetal calf serum. In the presence of cerebellar neurons, this rate was even higher. The specificity of the responsiveness of astrocytes to neurons with respect to glutamate uptake was investigated by comparing GABA uptake in the different culture conditions. Neurons also increased the rate of GABA uptake by astrocytes. Another component of the astroglial plasma membrane, the density of β‐adrenergic receptors, was, however, not markedly affected by the presence of neurons. Hence, these results showed that in astrocytes plated from postnatal day 4 mouse cerebella, the level of neuro‐transmitter uptake can be regulated in vitro by factors present in sera and by cerebellar neurons in the culture. However, this plasticity declined during development because astrocytes plated from postnatal day 8 cerebella and cultured under identical conditions were less active in glutamate uptake and were insensitive to the presence of horse serum. The latter observation suggested that the metabolic plasticity of astrocytes is restricted to a period defined early in cerebellar development and is no longer eviden

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1993.tb05829.x

出版商:Blackwell Publishing Ltd    

年代:1993

数据来源: WILEY

摘要:

Abstract:Generalised neurotransmitter overflow into the extracellular space on cerebral ischaemia has been widely reported and implicated in events leading to subsequent neu‐ronal death. As little is known about the effect of depth of ischaemia on these changes, we have subjected anaesthetised rats to a sequence of four challenges [high K+stimulus, moderate (penumbral) ischaemia, severe ischaemia, cardiac arrest] and have concurrently monitored both electrophysio‐logical parameters and changes in extracellular dopamine, serotonin, and their metabolites in the striatum. Oi'particu‐lar relevance to human stroke therapy was penumbral ischaemia, where ionic homeostasis was maintained even though electrical function was lost. All challenges increased extracellular monoamines, although levels were significantly greater when ischaemia was severe enough to produce sustained anoxic depolarisation. Baseline levels were rapidly restored during recovery phases. Acidic monoamine metabolites decreased significantly during each insult, returning to basal levels during reperfusion after moderate ischaemia, and to significantly higher levels after severe ischaemia. Results indicate that sustained anoxic depolarisation may be a critical factor in determining outcome after ischaemia, being associated with significantly greater release of monoamines, and impairment of electrical function rec

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1993.tb05830.x

出版商:Blackwell Publishing Ltd    

年代:1993

数据来源: WILEY

摘要:

Abstract:The effects of two new catechol‐O‐methyltransferase (COMT) inhibitors, OR‐611 and Ro 40‐7592, in combination with L‐3,4‐dihydroxyphenylalanine (L‐dopa) with or without carbidopa on extracellular levels of dopamine (DA), 3,4‐dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), 3‐O‐methyldopa (3‐OMD), and 5‐hydroxyindoleacetic acid in rat striatum were studied. A dose of 10 mg/kg i.p. of Ro 40‐7592 alone, in contrast to the same dose of OR‐611, decreased the dialysate level of HVA and increased that of DOPAC; this dose was thus used to differentiate between the effects of central and peripheral COMT inhibition. L‐Dopa (50 mg/kg i.p.) alone slightly increased extracellular levels of DA, DOPAC, and HVA. The effects of L‐dopa were potentiated by carbidopa (50 mg/kg i.p.), and even 3‐OMD levels in dialysate samples became detectable. Both OR‐611 and Ro 40‐7592 significantly further increased the DA and DOPAC efflux from striatum produced by L‐dopa. This increase was more pronounced when carbidopa was added to the treatment. OR‐611 did not modify the effect of L‐dopa or carbidopa/L‐dopa on dialysate HVA levels, whereas Ro 40‐7592 markedly reduced those levels. Both OR‐611 and Ro 40‐7592 very clearly suppressed dialysate 3‐OMD levels produced by carbidopa/L‐dopa. Ro 40‐7592 was more effective than OR‐611 in potentiating the effects of L‐dopa or carbidopa/L‐dopa. These in vivo data show that the new COMT inhibitors markedly inhibit theO‐methylation of L‐dopa and increase its availability to brain, which is reflected as increased DA formation. A significant effect can be achieved even by inhibiting only the peripheral COMT activity. The data suggest that COMT inhibitors may be of clin

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1993.tb05831.x

出版商:Blackwell Publishing Ltd    

年代:1993

数据来源: WILEY

摘要:

Abstract:The intracellular mechanisms through which two trophic factors, ciliary neurotrophic factor (CNTF) and leukemia inhibitory factor (LIF), regulate cholinergic development were examined in sympathetic neuron cultures. Treatment with CNTF or LIF increased levels of choline acetyltransferase (ChAT) activity by 375 and 350%, respectively. However, in neuronal cultures depleted of protein kinase C (PKC) activity by chronic phorbol ester treatment, neither CNTF nor LIF elevated ChAT activity. Further, the stimulation of ChAT due to increased cell density was not observed in PKC‐depleted sympathetic neurons. The inhibition of CNTF‐stimulated ChAT by phorbol ester occurred in a dose‐dependent manner and chronic phorbol ester treatments did not alter the levels of the catecholamine biosynthetic enzyme tyrosine hydroxylase. Moreover, increased levels of diacylglycerol, an endogenous activator of PKC, were observed in sympathetic neurons treated with CNTF. However, neither CNTF nor LIF stimulated the hydrolysis of phosphatidylinositol 4,5‐bisphosphate. These observations suggest that a common PKC‐dependent pathway, which is independent of phosphatidylinositol 4,5‐bisphosphate hydrolysis, mediates the cholinergic stimulating effects of CNTF, LIF, and cell‐cell contact in cultured sympat

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1993.tb05832.x

出版商:Blackwell Publishing Ltd    

年代:1993

数据来源: WILEY

摘要:

Abstract:Myelin basic protein (MBP) from common goldfish (Carassius auratus) myelin was extracted with dilute mineral acid. Immunological cross‐reactivity of the goldfish MBP, with polyclonal antisera raised against bovine MBP, suggested that the goldfish protein has epitopes for these antibodies. It also reacted with a monoclonal antibody specific for a seven amino acid epitope (130–137) conserved in the MBP of most mammalian species. To characterize the charge heterogeneity of this protein, we iodinated the protein with125I and chromatographed it on a carboxymethyl cellulose‐52 column together with a nonlabeled acid soluble fraction prepared from human white matter as a carrier protein. All of the goldfish protein was recovered in the unbound fraction, demonstrating that it was less cationic than the carrier protein (human MBP). We have also examined the urea alkaline gel profile of the goldfish MBP together with the human C‐1, C‐2, C‐3, C‐4, and C‐8 components. The results from these experiments indicated that this MBP extracted from goldfish brain myelin lacked the microhet‐erogeneity that is associated with MBPs from higher vertebrates. The MBPs from goldfish myelin were separated into their isoforms by reversed‐phase HPLC. Amino acid compositions were determined for both the 17‐ and 14‐kDa goldfish proteins. Amino acid analysis revealed similarities with the compositions of other MBPs; however, the serine content in both the 17‐ and 14‐kDa proteins was higher than that of the human C‐1, the mouse C‐1 protein, and the shark proteins. The HPLC‐purified 14‐kDa goldfish protein was chemically cleaved with CNBr for partial sequence analysis. Even from the limited sequence obtained, the sequence ATAST was found in goldfish, which is also present in

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1993.tb05833.x

出版商:Blackwell Publishing Ltd    

年代:1993

数据来源: WILEY

摘要:

Abstract:The effect of graded levels of tissue hypoxia on the extracellular levels of dopamine, 3,4‐dihydroxyphenylacetic acid, homovanillic acid, and 5‐hydroxyindoleacetic acid has been monitored in vivo by microdialysis. Reproducible levels of decreased oxygen in the brain were obtained by increasing the rate of ventilation from the control value of 25/min to as high as 95/min. With increasing ventilatory rate, the oxygen pressure in the cortex decreased from ∼40 torr to 16 torr. As the oxygen pressure decreased stepwise from 40 to 27, 22, and 16 torr, the dopamine levels in the extracellular medium rose by 70, 90, and 150%, respectively, returning to baseline within a few minutes of return to control ventilation rates. Levels of the catabolic products 3,4‐dihydroxyphenylacetic acid, homovanillic acid, and 5‐hydroxyindoleacetic acid decreased with decreasing tissue oxygen. Unlike the dopamine levels, these catabolite levels continued to decrease through 30 min of recovery (to 50% of control), returning to baseline only after recovery periods of 1–2 h. These data suggest that hypoxia induces long‐term alterations in the neurotransmitter turnover. The marked effects of mild tissue hypoxia (decrease of oxygen from 40 torr to 26 torr) on both the extracellular dopamine concentration and dopamine metabolism indicate that the metabolic consequences of decreased tissue oxygen pressure extend to higher values than generall

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1993.tb05834.x

出版商:Blackwell Publishing Ltd    

年代:1993

数据来源: WILEY

摘要:

Abstract:Some data suggest that the sodium‐dependent, high‐affinity L‐glutamate (Glu) transport sites in forebrain are different from those in cerebellum. In the present study, sodium‐dependent transport of L‐[3H]Glu was characterized in cerebellum and cortex. In both cerebellar and cortical tissue, activity was enriched in synaptosomes. Approximately 100 excitatory amino acid analogues were tested as potential inhibitors of transport activity. Many of the compounds tested inhibited transport activity by<65% at 1 mMand were not studied further. One group of compounds exhibited inhibition conforming to theoretical curves with Hill coefficients of 1 and were10‐fold selective as inhibitors. These included β‐N‐oxalyl‐L‐α,β‐diaminopropionate, α‐methyl‐DL‐glutamate, (2S, 1′S,2′S)‐2‐(carboxycyclopropyl)glycine, and (2S, 1′S,2′S,3′S)‐2‐(2‐carboxy‐3‐methoxymethylcyclopropyl)glycine. Data obtained with a few of the inhibitors were consistent with two sites in one or both of the brain regions. (2S, 1′R,2′R)‐2‐(Carboxycyclopropyl)glycine (L‐CCG‐II) was identified as the most potent (IC50= 5.5 μM) and selective (60–100‐fold) inhibitor of transport activity in cerebellum. One of the potential endogenous substrates, L‐homocysteate, was also a selective inhibitor of cerebellar transport activity. The data for inhibition of transport activity in cortex by both L‐CCG‐II and L‐homocysteate were best fit to two sites. Kainate was equipotent as an inhibitor of transport activity, and in both brain regions the data for inhibition were best fit to two sites. The possibility that there are four subtypes of excitatory amino acid transport is discussed. Altering sodium and potassium levels affects cerebellar and cortical transport activity differently,

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1993.tb05835.x

出版商:Blackwell Publishing Ltd    

年代:1993

数据来源: WILEY