摘要:

Abstract:A partial cDNA encoding most of the third intracellular loop of the mouse α1d‐adrenergic receptor subtype was amplified from hippocampus by reverse transcription‐polymerase chain reaction (RT‐PCR) using degenerate oligodeoxynucleotide primers. This DNA fragment was used as a probe to isolate an α1d‐adrenergic receptor cDNA from a mouse brain cDNA library. The deduced amino acid sequence encodes a potential protein of 562 amino acids, and northern hybridization of poly(A)+RNA isolated from mouse brain detected a single 3.0‐kb transcript. Partial cDNA fragments of the α1b‐ and α1a‐adrenergic receptor subtypes were also amplified from mouse brain and sequenced. Analysis of the mRNA expression by RT‐PCR indicated that the α1‐adrenergic receptors are widely distributed in mouse tissues. The α1dsubtype is expressed in brain areas such as hippocampus, striatum, and brainstem and also in many extracerebral tissues, such as lung, liver, heart, kidney, and spleen. The α1asubtype is also expressed in many tissues, whereas the α1bsubtype has a more restricted expression, with high levels in striatum, br

ISSN:0022-3042    

DOI:10.1046/j.1471-4159.1995.65062387.x

出版商:Blackwell Science Ltd    

年代:1995

数据来源: WILEY

摘要:

Abstract:A full‐length cDNA clone encoding for the chick CNTFRα (α component of the ciliary neurotrophic factor receptor) was isolated by screening an embryonic day 13 chick brain cDNA library with a rat CNTFRα probe. The isolated cDNA clone contained a ∼2‐kb insert with an open reading frame of 362 amino acids. The identification of this clone as chick CNTFRα was based on the homology in amino acid sequence (∼70%) with the rat and human CNTFRα. Hydropathy analysis revealed that the chick CNTFRα contains a hydrophobic region at the amino terminus that is typical of secretory signal peptides, as well as a hydrophobic region at the carboxyl terminus that is characteristic of glycosylphosphatidylinositol‐linked proteins. The expression of chick CNTFRα was developmentally regulated and was widely distributed in neural tissues, such as brain and spinal cord. In the periphery, chick CNTFRα transcript was expressed at high levels in the skeletal muscle and was only barely detectable in the liver. Unexpectedly, the expression of chick CNTFRα mRNA in skeletal muscle was decreased by ∼10‐fold at 1.5 days after denervation. This is in sharp contrast to the result previously obtained with CNTFRα i

ISSN:0022-3042    

DOI:10.1046/j.1471-4159.1995.65062393.x

出版商:Blackwell Science Ltd    

年代:1995

数据来源: WILEY

摘要:

Abstract:The alcohol‐sensitive (ANT) rat line, developed for high behavioral sensitivity to ethanol, also exhibits enhanced sensitivity to benzodiazepines, such as diazepam. The rat line carries a point mutation in the cerebellum‐specific γ‐aminobutyric acid type A (GABAA) receptor subunit α6, making their diazepam‐insensitive (DIS) receptors sensitive to diazepam. We now report that phenotypes of individual ANT and alcohol‐insensitive rats, classified on diazepam sensitivity of cerebellar [3H]Ro 15‐4513 binding, correlated well with homozygous wild‐type, homozygous mutant, and heterozygous genotypes, although some heterozygotes were biased toward the parental phenotypes. GABA down‐modulated DIS [3H]Ro 15‐4513 binding in mutant homozygotes but tended to up‐modulate it in heterozygotes and wild‐type homozygotes. Slopes for GABA inhibition of cerebellart‐butylbicyclophosphoro[35S]thionate binding were larger in mutant than in wild‐type homozygotes, with heterozygotes being intermediate. Diazepam displacement of [3H]Ro 15‐4513 binding in heterozygotes revealed three components, with their affinities indistinguishable from those in combined wild‐type and mutant homozygotes. This lack of interaction in DIS binding between wild‐type and mutant α6 subunits was substantiated by experiments on recombinant receptors. The data suggest that the α6 subunit‐containing GABAAreceptors in the heterozygotes are formed from individual mutant and wild‐type subunits with their relative exp

ISSN:0022-3042    

DOI:10.1046/j.1471-4159.1995.65062401.x

出版商:Blackwell Science Ltd    

年代:1995

数据来源: WILEY

摘要:

Abstract:Aromaticl‐amino acid decarboxylase (AADC) is expressed in a wide variety of tissues, including those where it is known to convertl‐DOPA and 5‐hydroxytryptophan to dopamine and serotonin, respectively. AADC has been cloned from many species and shown to undergo alternative splicing within its 5′ untranslated region. Here, we report that the human AADC gene can undergo additional alternative splicing of exon 3, generating two different protein isoforms (termed AADC480and AADC442). Both transcripts are widely expressed, with AADC442predominating in many neuronal and nonneuronal tissues. When homogenates were prepared from COS‐7 cells transfected with expression vectors containing either cDNA, AADC480catalyzed the decarboxylation of bothl‐DOPA and 5‐hydroxytryptophan. AADC442was inactive in either assay. These findings suggested that AADC442may have a different function in non‐monoamine‐expressing tissues. Taken together, these results suggest that the human AADC gene undergoes complex processing, leading to the formation of both tissue‐specific transcripts as well as uniq

ISSN:0022-3042    

DOI:10.1046/j.1471-4159.1995.65062409.x

出版商:Blackwell Science Ltd    

年代:1995

数据来源: WILEY

摘要:

Abstract:Among various tissues of mouse, β1,4‐N‐acetylgalactosaminyltransferase (GM2/GD2 synthase) gene is expressed predominantly in the brain. Further analysis of the gene expression in the mouse CNS was performed by northern blotting and by enzyme assays using extracts from various parts of the CNS. In situ hybridization was also done to investigate the distribution of cells generating GM2/GD2 synthase. In northern blots, diverse levels of the gene expression were observed, depending on the regions examined. By in situ hybridization, pyramidal cells in the hippocampus, granular cells in dentate gyrus and cerebral cortex, Purkinje cells in cerebellum, and mitral cells in the olfactory bulb expressed high levels of the mRNA; these results corresponded to the results obtained by northern blot. Enzyme levels in these sites were accordingly high. However, enzyme levels in certain areas with low mRNA intensities, such as thalamus and pons medulla, were higher than expected from the results of northern blotting. The significance of the high gene expression in certain areas for brain function and the reason for the discrepancy between mRNA level and enzyme activity in some regions are discu

ISSN:0022-3042    

DOI:10.1046/j.1471-4159.1995.65062417.x

出版商:Blackwell Science Ltd    

年代:1995

数据来源: WILEY

摘要:

Abstract:Cultured cerebellar granule neurons maintained in depolarizing concentrations of K+(25 mM) and then switched to physiological concentrations of K+(5 mM) undergo apoptosis. We now report that activation of specific G proteins robustly and bidirectionally affects apoptosis of cultured rat cerebellar granule neurons. Stimulation of Gswith cholera toxin completely blocks apoptosis induced by nondepolarizing concentrations of K+, whereas stimulation of Go/Giwith the wasp venom peptide mastoparan induces apoptosis of cerebellar granule neurons even in high (depolarizing) concentrations of K+. Moreover, pretreatment of cerebellar granule neurons with cholera toxin attenuates neuronal death induced by mastoparan. By contrast, pertussis toxin, cell‐permeable analogues of cyclic AMP, and activators of protein kinase A do not affect apoptosis of cultured cerebellar granule neurons. These data suggest that G proteins may function as key switches for controlling the programmed death of mammalian neurons, especially in the developing CN

ISSN:0022-3042    

DOI:10.1046/j.1471-4159.1995.65062425.x

出版商:Blackwell Science Ltd    

年代:1995

数据来源: WILEY

摘要:

Abstract:The protooncogenebcl‐2rescues cells from a wide variety of insults. Recent evidence suggests that the mechanism of action of Bcl‐2 involves antioxidant activity. The involvement of free radicals in ischemia/reperfusion injury to neural cells has led us to investigate the effect of Bcl‐2 in a model of delayed neural cell death. We have examined the survival of control andbcl‐2transfectants of a hypothalamic tumor cell line, GT1‐7, exposed to potassium cyanide in the absence of glucose (chemical hypoxia/aglycemia). After 30 min of treatment, no loss of viability was evident in control orbcl‐2transfectants; however, Bcl‐2‐expressing cells were protected from delayed cell death measured following 24–72 h of reoxygenation. Under these conditions, the rate and extent of ATP depletion in response to treatment with cyanide in the absence of glucose and the rate of recovery of ATP during reenergization were similar in control and Bcl‐2‐expressing cells. Bcl‐2‐expressing cells were protected from oxidative damage resulting from this treatment, as indicated by significantly lower levels of oxidized lipids. Mitochondrial respiration in control but not Bcl‐2‐expressing cells was compromised immediately following hypoxic treatment. These results indicate that Bcl‐2 can protect neural cells from delayed death resulting from chemical hypoxia and reenergization, and may do so by an antioxidant mechanism. The results thereby provide evidence that Bcl‐2 or a Bcl‐2 mimetic has potential therapeutic application in the treatment of neuropathologies involving oxidative stress, including

ISSN:0022-3042    

DOI:10.1046/j.1471-4159.1995.65062432.x

出版商:Blackwell Science Ltd    

年代:1995

数据来源: WILEY

摘要:

Abstract:VGF is a neuroendocrine‐specific gene product that is up‐regulated by nerve growth factor in the PC12 cell line. In rat neuroendocrine tissues two polypeptides of 90 and 80 kDa were detected by an antiserum to an N‐terminal domain of VGF (from residues 4 to 240). In parallel, an antiserum directed against the C‐terminal nonapeptide of VGF (from residues 609 to 617) revealed several additional posttranslational products. Peptides of apparent molecular sizes of 20, 18, and 10 kDa were prominent in nerve tissues and the hypophysis but absent in the adrenal medulla, and their relative abundance varied in distinct regions of the CNS. In PC12 cells VGF was proteolytically processed only after nerve growth factor treatment, and primary cultures of rat cerebellar granule cells accumulated the low‐molecular‐weight forms of VGF during in vitro maturation. In these cells the specific cleavages of VGF occurred in a postendoplasmic reticulum compartment; the processed forms were enriched in the secretory vesicles and were preferentially secreted upon cell membrane depolarization. Distinct differential distribution in the CNS and in vitro release of such posttranslational products indicate that these species may represent biologically relevant forms of VGF that play a role in neuronal co

ISSN:0022-3042    

DOI:10.1046/j.1471-4159.1995.65062441.x

出版商:Blackwell Science Ltd    

年代:1995

数据来源: WILEY

摘要:

Abstract:To elucidate the significance of branched‐chain amino acids (BCAAs) for brain energy metabolism, the capacity to use BCAAs for oxidative metabolism was investigated in astroglia‐rich primary cultures derived from newborn rat brain. The cells selectively removed BCAAs from the culture medium, the disappearance following first‐order kinetics. The BCAAs disappeared rapidly in spite of the presence of sufficient glucose as substrate for the generation of energy. Taking into consideration that the ketogenic amino acid leucine could be degraded only to acetyl‐CoA and acetoacetate, and with the knowledge that astroglial cells have the capacity to secrete ketone bodies, this amino acid was chosen for further metabolic studies. After incubation of the cells with leucine, acetoacetate,d‐β‐hydroxybutyrate, and α‐ketoisocaproate were found to have accumulated in the culture medium. Identification of the radioactive metabolites generated from [4,5‐3H]leucine established that the source of the substances released was indeed leucine. These results indicate that, at least in culture, astroglial cells degrade leucine via the known metabolite α‐ketoisocaproate, to acetoacetate, which can be further reduced tod‐β‐hydroxybutyrate. It is hypothesized that upon release from brain astrocytes, the ketone bodies could serve as fuel molecules for neighboring cells such as neurons and oligodendrocytes. In view of these and other results, astrocytes may be considered the brain

ISSN:0022-3042    

DOI:10.1046/j.1471-4159.1995.65062450.x

出版商:Blackwell Science Ltd    

年代:1995

数据来源: WILEY

摘要:

Abstract:Pro‐thyrotropin‐releasing hormone (proTRH) is the precursor to thyrotropin‐releasing hormone (TRH; pGlu‐His‐Pro‐NH2), the hypothalamic releasing factor that stimulates synthesis and release of thyrotropin from the pituitary gland. Five copies of the TRH progenitor sequence (Gln‐His‐Pro‐Gly) and seven cryptic peptides are formed following posttranslational proteolytic cleavage of the 26‐kDa rat proTRH precursor. The endopeptidase(s) responsible for the physiological conversion of proTRH to the TRH progenitor form is currently unknown. We examined the in vitro processing of [3H]leucine‐labeled or unlabeled proTRH by partially purified recombinant PC1. Recombinant PC1 processed the 26‐kDa TRH precursor by initially cleaving the prohormone after the basic amino acid at either position 153 or 159. Based on the use of our well‐established antibodies, we propose that the initial cleavage gave rise to the formation of a 15‐kDa N‐terminal peptide (preproTRH25–152or preproTRH25–158) and a 10‐kDa C‐terminal peptide (preproTRH154–255or preproTRH160–255). Some initial cleavage occurred after amino acid 108 to generate a 16.5‐kDa C‐terminal peptide. The 15‐kDa N‐terminal intermediate was further processed to a 6‐kDa peptide (preproTRH25–76or preproTRH25–82) and a 3.8‐kDa peptide (preproTRH83–108), whereas the 10‐kDa C‐terminal intermediate was processed to a 5.4‐kDa peptide (preproTRH206–255). The optimal pH for these cleavages was 5.5. ZnCl2, EDTA, EGTA, and the omission of Ca2+inhibited the formation of pYE27(preproTRH25–50), one of the proTRH N‐terminal products, by 48, 82, 72, and 45%, respectively. This study provides evidence, for the first time, that recombinan

ISSN:0022-3042    

DOI:10.1046/j.1471-4159.1995.65062462.x

出版商:Blackwell Science Ltd    

年代:1995

数据来源: WILEY