ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1992.tb11385.x

出版商:Blackwell Publishing Ltd    

年代:1992

数据来源: WILEY

摘要:

AbstractThe extracellular matrix (ECM) at the vertebrate neuromuscular junction is a repository of functionally important molecules, some of which can regulate the formation of synapses during regeneration. One candidate molecule is s‐laminin, a 185‐kDa homologue of the laminin B1 chain. Whereas several members of the laminin family are present throughout the ECM ensheathing muscle fibers, immunoreactivity for s‐laminin is found selectively at synaptic sites in adult and embryonic rats, and is detectable at a time when synaptogenesis is taking place during development. We have reported previously that a rat schwannoma cell line, D6P2T, produces and releases large amounts of s‐laminin in culture. We have now purified s‐laminin from medium conditioned by these cells by using a simple three‐step procedure. Serum‐free, conditioned medium is separated by ion‐exchange chromatography on DEAE‐Sephacel, followed by size‐exclusion chromatography on 500 HR‐Seph‐acryl. Finally, s‐laminin is dissociated from other ECM components by agarose gel electrophoresis under reducing conditions and recovered in solution by extracting slices of agarose gel. The purified preparation displays one silver‐stained band that is recognized by three monoclonal antibodies known to bind to different epitopes on s‐laminin. Lectin‐binding studies demonstrate that s‐laminin is a glycoprotein and bears many of the carbohydrate moieties present on the B1 and B2 chains of laminin. Thus, the three 185–220‐kDa members of the laminin family are related in both t

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1992.tb08869.x

出版商:Blackwell Publishing Ltd    

年代:1992

数据来源: WILEY

摘要:

AbstractThe rate of leucine incorporation into brain proteins was studied in rats with experimental brain tumors produced by intracerebral transplantation of the glioma clone F98. Incorporation was measured with [14C]leucine using a controlled infusion technique for maintaining constant specific activity of [14C]leucine in plasma, followed by quantitative autoradiography and biochemical tissue analysis. After 45 min the specific activity of free [14C]leucine in plasma was 2.5–3 times higher than in brain and brain tumor, indicating that the precursor pool for protein synthesis was fueled both by exogenous (plasma‐derived) and endogenous (proteolysis‐derived) amino acids. Endogenous recycling of amino acids amounted to 73% of total free leucine pool in brain tumors and to 60–70% in normal brain. Taking endogenous amino acid recycling into account, leucine incorporation was 78.7 ± 16.0 nmol/g of tissue/min in brain tumor, and 17.2 ± 4.2 and 9.7 ± 3.3 nmol/g/min in normal frontal cortex and striatum, respectively. Leucine incorporation within tumor tissue was markedly heterogeneous, depending on the local pattern of tumor proliferation and necrosis. Our results demonstrate that quantitative measurement of leucine incorporation into brain proteins requires estimation of recycling of amino acids derived from proteolysis and, in consequence, biochemical determination of the free amino acid precursor pool in tissue samples. With the present approach such measurements are possible and provide the quantitative basis for the evaluation of therapeutic int

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1992.tb08870.x

出版商:Blackwell Publishing Ltd    

年代:1992

数据来源: WILEY

摘要:

AbstractProduction of active enkephalin peptides requires proteolytic processing of proenkephalin at dibasic Lys‐Arg, Arg‐Arg, and Lys‐Lys sites, as well as cleavage at a monobasic arginine site. A novel “prohormone thiol protease” (PTP) has been demonstrated to be involved in enkephalin precursor processing. To find if PTP is capable of cleaving all the putative cleavage sites needed for proenkephalin processing, its ability to cleave the dibasic and the monobasic sites within the enkephalin‐containing peptides, peptide E and BAM‐22P (bovine adrenal medulla docosapeptide), was examined in this study. Cleavage products were separated by HPLC and subjected to microsequencing to determine their identity. PTP cleaved BAM‐22P at the Lys‐Arg site between the two basic residues. The Arg‐Arg site of both peptide E and BAM‐22P was cleaved at the NH2‐terminal side of the paired basic residues to generate [Met]‐enkephalin. Furthermore, the monobasic arginine site was cleaved at its NH2‐terminal side by PTP. These findings, together with previous results showing PTP cleavage at the Lys‐Lys site of peptide F, demonstrate that PTP possesses the necessary specificity for all the dibasic and monobasic cleavage sites required for proenkephalin processing. In addition, the unique specificity of PTP for cleavage at the NH2‐terminal side of arginine at dibasic or monobasic sites distinguishes it from many other putative prohormone processing enzymes, providing further evidence that PTP appears to be a nov

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1992.tb08871.x

出版商:Blackwell Publishing Ltd    

年代:1992

数据来源: WILEY

摘要:

AbstractThe metabolism of lactate in isolated cells from early neonatal rat brain has been studied. In these circumstances, lactate was mainly oxidized to CO2, although a significant portion was incorporated into lipids (78% sterols, 4% phosphatidylcholine, 2% phosphatidylethanolamine, and 1% phosphatidylserine). The rate of lactate incorporation into CO2and lipids was higher than those found for glucose and 3‐hydroxybutyrate. Lactate strongly inhibited glucose oxidation through the pyruvate dehydrogenase‐catalyzed reaction and the tricarboxylic acid cycle while scarcely affecting glucose utilization by the pentose phosphate pathway. Lipogenesis from glucose was strongly inhibited by lactate without relevant changes in the rate of glycerol phosphate synthesis. These results suggest that lactate inhibits glucose utilization at the level of the pyruvate dehydrogenase‐catalyzed reaction, which may be a mechanism to spare glucose for glycerol and NADPH synthesis. The effect of 3‐hydroxybutyrate inhibiting lactate utilization only at high concentrations of 3‐hydroxybutyrate suggests that before ketogenesis becomes active, lactate may be the major fuel for the neonatal brain. (‐)‐Hydroxycitrate and aminooxyacetate markedly inhibited lipogenesis from lactate, suggesting that the transfer of lactate carbons through the mitochondrial membrane is accomplished by the translocation of both citrate andN‐

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1992.tb08872.x

出版商:Blackwell Publishing Ltd    

年代:1992

数据来源: WILEY

摘要:

AbstractSimultaneous treatment with 12‐O‐tetradecanoylphorbol 13‐acetate (TPA) and dibutyryl cyclic AMP (diBu‐cAMP) for 72 h induced neurites in NG108–15 cells significantly longer than treatment with each alone. Treatment for 72 h with both drugs induced irreversible neurite extension and a decline in protein kinase C activity, although neurites extended by diBu‐cAMP alone disappeared after the withdrawal of the drug. The expression of growth‐associated protein‐43 (GAP‐43) mRNA was also observed by a combined application of TPA and diBu‐cAMP. The increased level of GAP‐43 mRNA induced by treatment with both drugs for 72 h was maintained at least 24 h after withdrawal of the drugs. In cells transfected with GAP‐43 cDNA, neurites induced by treatment with diBu‐cAMP alone for 72 h were maintained at least 48 h after removal of the drugs. These results suggest that GAP‐43 could be involved in the maintenance of elongated neurites and that a decline in protein kinase C activity may be involved in

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1992.tb08873.x

出版商:Blackwell Publishing Ltd    

年代:1992

数据来源: WILEY

摘要:

AbstractWe examined the effect of chronic nicotine treatment on dopaminergic activity by measuring the effects of D1and D2dopamine (DA) receptor agonists and antagonists on tritium release from mouse striatum preloaded with [3H]DA. The radioactivity released during superfusion was separated on alumina columns and the distribution and efflux of [3H]DA and its main3H‐labeled metabolites were quantified. After preloading by incubation with [3H]DA, the electrical stimulation‐evoked tritium overflow was higher in striatum prepared from nicotine‐treated mice, whereas in vitro addition of nicotine caused a similar increase in tritium release from striatum of untreated and chronic nicotine‐treated mice. The overflow of [3H]DA and its3H‐metabolites exhibited similar distribution patterns in [3H]DA‐preloaded striatum dissected from untreated and chronic nicotine‐pretreated mice, indicating that repeated injections with nicotine did not alter the metabolism of [3H]DA taken up by the tissue. (−)‐Quinpirole, a selective agonist for D2DA receptors, and apomorphine, a nonselective D1/D2agonist, inhibited the electrical stimulation‐induced tritium efflux from striatum of untreated mice, whereas (±)‐sulpiride, a D2DA receptor antagonist, enhanced the evoked release of tritium. These changes in tritium efflux effected by (−)‐quinpirole and (±)‐sulpiride reflected changes in [3H]DA release and not in DA metabolism, as shown by separation of the released radioactivity on alumina columns. The D1receptor agonist (±)‐SKF‐38393 did not affect the tritium overflow, whereas the D1receptor antagonist (+)‐SCH‐23390 exerted a stimulatory action but only at a high concentration. In contrast, neither the DA receptor agonists [(−)‐quinpirole, apomorphine, and (±)‐SKF‐38393] nor the antagonists [(±)‐sulpiride and (+)‐SCH‐23390]altered the stimulation‐evoked tritium release in striatum obtained from mice repeatedly treated with nicotine. It is concluded that chronic administration of nicotine produces an increased release of DA in the striatum with a resulting elevation of synaptic DA concentrations leading to development of D2DA autoreceptor subsensitivity. As a consequence, chronic nicotine may attenuate autoinhi

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1992.tb08874.x

出版商:Blackwell Publishing Ltd    

年代:1992

数据来源: WILEY

摘要:

AbstractTo test the specificity ofN‐acetylaspartate (NAA) as a neuronal marker for proton nuclear magnetic resonance (1H NMR) spectroscopy, purified and characterized cultured cells were analyzed for their NAA content using both1H NMR and HPLC. Cell types studied included cerebellar granule neurons, type‐1 astrocytes, meningeal cells, oligodendrocyte‐type‐2 astrocyte (O–2A) progenitor cells, and oligodendrocytes. A high concentration of NAA was found in extracts of cerebellar granule neurons (approximately 12 nmol/mg of protein), whereas NAA remained undetectable in purified type‐1 astrocytes, meningeal cells, and mature oligodendrocytes. However, twice the neuronal level of NAA was found in O‐2A progenitors grown in vitro. In addition significant levels of NAA were also detected in cultures of immature oligodendrocytes. Our data partly support previous suggestions that NAA may be a useful neuronal marker for1H NMR spectroscopic examination of the adult brain. However, they also raise the further possibility that alterations of NAA associated with some specific brain disorders, particularly disorders seen in newborn and young children, may reflect abnormalities in the development of oligodendroglia or the

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1992.tb08875.x

出版商:Blackwell Publishing Ltd    

年代:1992

数据来源: WILEY

摘要:

AbstractWe have quantitated the α1, α5, γ2S, and γ2Lγ‐aminobutyric acidA(GABAA) receptor subunit mRNAs in the maturing cerebellum in vivo and in cerebellar granule neurons differentiating in vitro. Absolute amounts of mRNA were measured by reverse transcription and competitive polymerase chain reaction (PCR) analysis with appropriate internal standards. The α1and γ2LmRNA content increased continuously during postnatal cerebellar maturation and their changes with time matched very closely those of the cerebellar granule cells differentiating in vitro. The γ2Ssubunit mRNA showed a relatively constant pattern of expression both in vivo and in vitro, with comparable absolute concentrations in both developmental paradigms. The α5mRNA was initially high in vivo and decreased (eightfold) to adult levels as postnatal cerebellar development progressed. In vitro the amount of α5GABAAreceptor subunit mRNA was higher than in vivo at 3 days, increased by more than twofold by 8 days, and declined to approximately the initial values at 23 and 28 days in vitro. Collectively, the results indicate that the α1, α5, γ2S, and γ2LGABAAreceptor subunit mRNAs are regulated differentially in a temporal manner during in vivo and in vitro maturation. Moreover, a comparison of the ontogenetic profiles of the γ2Sand γ2LmRNAs indicates that alternative splicing of the γ2primary RNA transcript is regulated developmentally during postnatal maturation of

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1992.tb08876.x

出版商:Blackwell Publishing Ltd    

年代:1992

数据来源: WILEY

摘要:

AbstractNeuropeptide Y (NPY) was measured in postmortem brain tissue from victims of suicide and from individuals dying a sudden natural or accidental death (controls). Concentrations of NPY‐immunoreactivity were measured by radioimmunoassay in frontal cortex (BA 10), temporal cortex (BA 22), caudate nucleus, and cerebellum. Concentrations of NPY‐immunoreactivity were significantly lower in postmortem frontal cortex (−14%) and caudate nucleus (−27%) from suicide victims compared with age‐matched controls. A subgroup of suicides with evidence of a history of depression revealed more robust reductions in concentrations of NPY‐immunoreactivity in frontal cortex and caudate nucleus, as did four individuals who died from natural causes and also were described as having a possible history of depression. Concentrations of NPY‐immunoreactivity in temporal cortex and cerebellum from victims of suicide or from the subgroup of subjects with a possible history of depression were not significantly different from those of age‐matched controls. We suggest there is a deficit in the brain NPY system leading to region‐specific reductions in peptide concentrations in subjects who have a hist

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1992.tb08877.x

出版商:Blackwell Publishing Ltd    

年代:1992

数据来源: WILEY