摘要:

Abstract:It is well known that the regulation of choline acetyltransferase (ChAT) activity under physiological and pathological conditions is important for the development and neuronal activities of cholinergic systems involved in many fundamental brain functions. This review focuses on recent progress in understanding the regulation of ChAT at the levels of both the protein and the mRNA. A deficiency in ChAT activity has been reported for neurodegenerative conditions such as Alzheimer's disease, amyotrophic lateral sclerosis, and schizophrenia. Although a major feature of ChAT regulation is likely to involve the spatial and temporal control of transcription, regulation of expression can also be at the level of RNA processing, transport/ translocation, turnover, or translation. In addition, there is increasing evidence that ChAT might be regulated at the posttranslational level by compartmentation and/or covalent modification, i.e., phosphorylation, as well as noncovalent modification (protein‐protein interaction, etc.). Synaptic activity and the state of neuronal transmission may also involve the regulation of ChAT at different levels via both positive and negative feedback loops, as was demonstrated in the characterization of two ChAT mutantDrosophilastrains. Clearly, identification of cholinergic‐specific elements and the characterization of the trans‐acting factors that bind to them represent an important area of future research. Equally important is research on the mechanisms governing ChAT as an enzymatic entity. The future should be an exciting time during which we look forward to the elucidation of the cholinergic signal and its regulation as well as the determination of the three‐dimensional structure of the

ISSN:0022-3042    

DOI:10.1046/j.1471-4159.1994.62051653.x

出版商:Blackwell Publishing Ltd    

年代:1994

数据来源: WILEY

摘要:

Abstract:A histidine residue (His394) that is likely to be located in the ligand‐binding region of the D2dopamine receptor has been mutated to a leucine (Leu394), and the properties of the mutant receptor have been determined. For a range of antagonists the mutation has only a minor effect on the affinity of the receptor for the antagonist. The mutation does, however, elicit a structurally specific effect on the affinity with which certain members of the substituted benzamide class of antagonist bind to the receptor. Some of these drugs, e.g., sulpiride, sultopride, and tiapride, bind with reduced affinity to the mutated receptor, whereas others, e.g., clebopride and metoclopramide, bind with increased affinity. However, the Na+/H+sensitivity of the binding of sulpiride to the receptor is not reduced by the mutation. These findings have been interpreted in terms of the productive or unfavourable interaction of the His394residue with these compound

ISSN:0022-3042    

DOI:10.1046/j.1471-4159.1994.62051664.x

出版商:Blackwell Publishing Ltd    

年代:1994

数据来源: WILEY

摘要:

Abstract:Formation of a functional neuromuscular junction (NMJ) involves the biosynthesis and transport of numerous muscle‐specific proteins, among them the acetylcholine‐hydrolyzing enzyme acetylcholinesterase (AChE). To study the mechanisms underlying this process, we have expressed DMA encoding human AChE downstream of the cytomegalovirus promoter in oocytes and developing embryos ofXenopus laevis.Recombinant human AChE (rHAChE) produced inXenopuswas biochemically and immunochemically indistinguishable from native human AChE but clearly distinguished from the endogenous frog enzyme. In microinjected embryos, high levels of catalytically active rHAChE induced a transient state of over‐expression that persisted for at least 4 days postfertilization. rHAChE appeared exclusively as nonassembled monomers in embryos at times when endogenousXenopusAChE displayed complex oligomeric assembly. Nonetheless, cell‐associated rHAChE accumulated in myotomes of 2‐and 3‐day‐old embryos within the same sub‐cellular compartments as nativeXenopusAChE. NMJs from 3‐day‐old DNA‐injected embryos displayed fourfold or greater overexpression of AChE, a 30% increase in postsynaptic membrane length, and increased folding of the postsynaptic membrane. These findings indicate that an evolutionarily conserved property directs the intracellular trafficking and synaptic targeting of AChE in muscle and support a role for AChE in vert

ISSN:0022-3042    

DOI:10.1046/j.1471-4159.1994.62051670.x

出版商:Blackwell Publishing Ltd    

年代:1994

数据来源: WILEY

摘要:

Abstract:We report the isolation of cDNA clones encoding the somatic form of the E1α subunit of the pyruvate dehydrogenase complex of rat. The deduced amino acid sequence has 99.5, 98, and 97% identity, respectively, with the orthologous proteins of mouse, human, and pig and 98.5% identity with a rat E1α sequence reported previously. The cDNAs isolated in this and earlier studies predict different E1α subunit mRNA sizes and amino acid sequences. These differences have been investigated by PCR, northern blot hybridization, and RNase protection. We have used our E1α cDNA, in conjunction with cDNA probes to the E1β, E2, and E3 catalytic subunits of rat pyruvate dehydrogenase complex and also to rat citrate synthase, to perform RNase protection assays of developing rat whole brain RNA. The results show a 2.5‐fold increase in the concentration of each of the subunit mRNAs and a 1.2‐fold increase in citrate synthase mRNA from late foetal stage to 5 days post partum. Thereafter, the mRNA levels remained constant. These data indicate that the respective six‐and threefold increases in the amounts of pyruvate dehydrogenase complex and citrate synthase found to occur in rat brain between birth and adulthood are mediated principally by translational and/or posttranslational

ISSN:0022-3042    

DOI:10.1046/j.1471-4159.1994.62051682.x

出版商:Blackwell Publishing Ltd    

年代:1994

数据来源: WILEY

摘要:

Abstract:In order to define cell type‐specific elements associated with the catecholamine biosynthetic enzyme, tyrosine hydroxylase (TH), transient transfections of promoter deletion constructs were used to test relative reporter‐gene activities in TH‐expressing and‐nonexpressing cell lines. Such assays demonstrated that a region between‐503 and‐578 contributed to rat TH promoter activity in the pheochromocytoma cell line PC12. Deletion of these sequences resulted in a 66% loss in cell type‐specific activity. Mutations within the E box/dyad symmetry element (CAGGTGCCTGTGACAGTG) did not affect the basal and cell type‐specific pattern of expression exhibited by the rat TH promoter. Promoter fusion constructs between the rat TH promoter (‐741 and‐197) and the human TH promoter (‐197 and +1) exhibited reporter‐gene activities equivalent to that of wild‐type‐741 rat TH constructs, further demonstrating that sequence elements upstream of the rat E box/dyad symmetry are important for cell type‐specific expression. Gel‐shift experiments indicated that a PC12 nuclear factor could bind to a 39‐bp sequence within this region in a cell type‐specific manner. The size of this factor was 52 kDa as determi

ISSN:0022-3042    

DOI:10.1046/j.1471-4159.1994.62051691.x

出版商:Blackwell Publishing Ltd    

年代:1994

数据来源: WILEY

摘要:

Abstract:The relation between the availability of newly synthesized protein and lipid and the axonal transport of optically detectable organelles was examined in peripheral nerve preparations of amphibia (RanacatesbeianaandXenopus laevis)in which intracellular traffic from the endo‐plasmic reticulum to the Golgi complex was inhibited with brefeldin A (BFA). Accumulation of fast‐transported radio‐labeled protein or phospholipid proximal to a sciatic nerve ligature was monitored in vitro in preparations of dorsal root ganglia and sciatic nerve. Organelle transport was examined by computer‐enhanced video microscopy of single myelinated axons. BFA reduced the amount of radiolabeled protein and lipid entering the fast‐transport system of the axon without affecting either the synthesis or the transport rate of these molecules. The time course of the effect of BFA on axonal transport is consistent with an action at an early step in the intrasomal pathway, and with its action being related to the observed rapid (99%. The findings suggest that the anterograde flux of transport organelles is not critically dependent on a supply of newly synthesized membrane precursors. The possibilities are considered that anterograde organelles normally arise from membrane components supplied from a post‐Golgi storage pool, as well as from recycled retrogra

ISSN:0022-3042    

DOI:10.1046/j.1471-4159.1994.62051698.x

出版商:Blackwell Publishing Ltd    

年代:1994

数据来源: WILEY

摘要:

Abstract:Previous studies have demonstrated that bovine chromaffin cells cultured in medium with 10 nMinsulin‐like growth factor‐I (IGF‐I) secrete about twofold more catecholamine when exposed to secretory stimuli than do cells cultured without IGF‐I. The purpose of this study was to determine whether protein kinase C (PKC) is involved in the effect of IGF‐I on secretion from these cells. PKC was down‐regulated in the cells by 16–18 h of treatment with β‐phorbol didecanoate (β‐PDD; 100 nM). Such treatment had no effect on high‐K+‐stimulated secretion from cells cultured without IGF‐I; however, secretion from cells cultured with IGF‐I was reduced to a level comparable to that in cells cultured without the peptide. The inactive isomer, α‐PDD (100 nM), had no effect on secretion from untreated or IGF‐I‐treated chromaffin cells. The effect of β‐PDD was time and concentration dependent, with 100 nM β‐PDDproducing a maximal effect in 8–10 h. In situ PKC activity measured in permeabilized cells treated with PMA (300 nM) was decreased by∼40% by 10 h and was reduced to almost basal levels by 18 h. Immunoblotting experiments demonstrated that both α‐and ε‐PKC were lost from the cells with time courses similar to that seen in the in situ PKC assay. Overnight treatment with the PKC inhibitor H7 (100 μM) prevented the enhanced secretion normally seen in IGF‐l‐treated cells, whereas HA1004 had no effect. High‐K+‐stimulated45Ca2+uptake in IGF‐I‐treated cells was attenuated by long‐term treatment with β‐PDD (200 nM) or H7 (100 μM). Together these observations suggest th

ISSN:0022-3042    

DOI:10.1046/j.1471-4159.1994.62051707.x

出版商:Blackwell Publishing Ltd    

年代:1994

数据来源: WILEY

摘要:

Abstract:The nuclear factorKB(NF‐kB) is a eukaryotic transcription factor. In B cells and macrophages it is constitutively present in cell nuclei, whereas in many other cell types, NF‐KB translocates from cytosol to nucleus as a result of transduction by tumor necrosis factor α (TNFα), phorbol ester, and other polyclonal signals. Using neuro‐blastoma cell lines as models, we have shown that in neural cells NF‐KB was present in the cytosol and translocated into nuclei as a result of TNFa treatment. The TNFα‐activated NF‐KB was transcriptionally functional. NF‐KB activation by TNFα was not correlated with cell differentiation or proliferation. However, reagents such as nerve growth factor (NGF) and the phorbol ester phorbol 12‐myristate 13‐acetate (PMA), which induce phenotypical differentiation of the SH‐SY5Y neuroblastoma cell line, activated NF‐KB, but only in that particular cell line. In a NGF‐responsive rat pheochromocytoma cell line, PC12, PMA activated NF‐KB, whereas NGF did not. In other neuroblastoma cell lines, such as SK‐N‐Be(2), the lack of PMA induction of differentiation was correlated with the lack of NF‐kB activation. We found, moreover, that in SK‐N‐Be(2) cells protein kinase C (PKC) enzymatic activity was much lower compared with that in a control cell line and that the low PKC enzymatic activity was due to low PKC protein expression. NF‐KB was not activated by retinoic acid, which induced morphological differentiation of all the neuroblastoma cell lines used in the present study. Thus, NF‐KB activation was not required for neuroblastoma cell differentiation. Furthermore, the results obtained with TNFα proved thatNF‐KBactivation was not sufficient

ISSN:0022-3042    

DOI:10.1046/j.1471-4159.1994.62051716.x

出版商:Blackwell Publishing Ltd    

年代:1994

数据来源: WILEY

摘要:

Abstract:Synthesis, uptake, release, and oxidative metabolism of citrate were investigated in neurons and astrocytes cultured from cerebral cortex or cerebellum. In addition, the possible role of citrate as a donor of the carbon skeleton for biosynthesis of neurotransmitter glutamate was studied. All cell types expressed the enzyme citrate synthase at a high activity, the cerebellar granule neurons containing the enzyme at a higher activity than that found in the astrocytes from the two brain regions or the cortical neurons. Saturable citrate uptake could not be detected in any of the cell types, but the astrocytes, and, in particular, those of cerebellar origin, had a very active de novo synthesis and release of citrate (∼70 nmol × h−1× mg of protein−1). The rate of release of citrate from neurons was<5% of this value. Using [14C]citrate it could be shown that citrate was oxidatively metabolized to14CO2at a modest rate (∼1 nmol × n−1× mg−1of protein) with slightly higher rates in astrocytes compared with neurons. Experiments designed to investigate the ability of exogenously supplied citrate to serve as a precursor for synthesis of transmitter glutamate in cerebellar granule neurons failed to demonstrate this. Rather than citrate serving this purpose it may be suggested that astrocytically released citrate may regulate the extracellular concentration of Ca2+and Mg2+by chelation, thereby modulating neuron

ISSN:0022-3042    

DOI:10.1046/j.1471-4159.1994.62051727.x

出版商:Blackwell Publishing Ltd    

年代:1994

数据来源: WILEY

摘要:

Abstract:We applied in vivo microdialysis to assess the effects of dopaminergic and β‐adrenergic receptor stimulation on cyclic AMP efflux in rat striatum under chloral hydrate anesthesia. Dopamine (up to 1mM) infused for 20 min through the probe did not increase cyclic AMP, whereas both the selective dopamine D1agonist SKF 38393 and D2antagonist sulpiride produced modest increases. It is interesting that the β‐adrenoceptor agonist isoproterenol produced a marked increase (204.7% of basal level at 1 mM) which was antagonized by the β‐adreno‐ceptor antagonist propranolol. Pretreatment with a glial selective metabolic inhibitor, fluorocitrate (1mM), by a 5‐h infusion through the probe attenuated basal cyclic AMP efflux by 30.3% and significantly blocked the response to isoproterenol. By contrast, striatal injection of a neuro‐toxin, kainic acid (2.5 μg), 2 days before the dialysis experiment did not affect basal cyclic AMP or the response to isoproterenol, but blocked the response to SKF 38393. These data demonstrate that β‐adrenoceptors as well as dopamine receptors contribute to cyclic AMP efflux in rat striatum in vivo. They also suggest that basal and β‐adre‐noceptor‐stimulated cyclic AMP efflux are substantially depend

ISSN:0022-3042    

DOI:10.1046/j.1471-4159.1994.62051734.x

出版商:Blackwell Publishing Ltd    

年代:1994

数据来源: WILEY