摘要:

Abstract:DNA binding activities of a variety of transcription factors with different protein motifs were determined in nuclear extracts of mouse brains using radiolabeled double‐stranded oligonucleotides containing the respective consensus core elements as probes in gel‐retardation electrophoresis. DNA binding activities of the transcription factor with a leucine‐zipper motif activator protein 1 (AP1) were markedly modulated by the addition of endogenous monovalent and divalent cations at physiological concentrations. In the presence of both KCl and MgCl2at maximally effective concentrations, the AP1 binding occurred in a temperature‐dependent manner in brain nuclear extracts. Brain nuclear extracts also contained activities to bind probes for seven other transcription factors with leucine‐zipper, zinc finger, or helix‐turn‐helix motifs under the conditions favorable to detecting the AP1 binding. In contrast to brain nuclear extracts, however, both cations and incubation temperature were ineffective in markedly affecting binding of all eight radioprobes tested in hepatic nuclear extracts. Moreover, the addition of hepatic nuclear extracts eliminated the AP1 binding in brain extracts nearly completely, differentially affecting binding of other probes. Size‐exclusion chromatography on hepatic nuclear extracts revealed two distinct fractions with different molecular sizes that both have an activity to inhibit the AP1 binding in brain nuclear extracts. These results suggest that monovalent and divalent cations may modulate DNA binding activities of particular transcription factors in brain nuclear extracts but not in hepatic nuclear extracts that could contain high molecular weight materials with an inhibitory potency on brain DNA bin

ISSN:0022-3042    

DOI:10.1046/j.1471-4159.1995.64041431.x

出版商:Blackwell Science Ltd    

年代:1995

数据来源: WILEY

摘要:

Abstract:5‐Hydroxytryptamine (5‐HT) receptors contain seven putative transmembrane domains and couple via different guanine nucleotide binding proteins to specific effector enzymes. Studies with other receptors identify the second and third intracellular loops or the C‐terminus of the receptor as important for selective effector coupling. However, it is not known which regions of the 5‐HT receptor determine effector coupling specificity. To address this question, we constructed a chimeric 5‐HT receptor in which the third intracellular (i3) loop is derived from the 5‐HT2Areceptor, which is coupled to activation of phospholipase C, and the rest of the sequence is derived from the 5‐HT1Breceptor, which is coupled to inhibition of adenylyl cyclase. The chimeric receptor exhibited ligand binding properties similar to those of the 5‐HT1Breceptor and distinct from those of the 5‐HT2Areceptor. This suggests that the i3 loop is not critical for the unique pharmacology of the 5‐HT1Breceptor. In contrast, the chimeric receptor exhibited signaling properties similar to those of the 5‐HT2Areceptor and distinct from those of the 5‐HT1Breceptor. This indicates that the i3 loop determines the effector coupling specificit

ISSN:0022-3042    

DOI:10.1046/j.1471-4159.1995.64041440.x

出版商:Blackwell Science Ltd    

年代:1995

数据来源: WILEY

摘要:

Abstract:In this study, we describe the cloning and characterization of a soluble form of kynurenine aminotransferase (KAT, EC 2.6.1.7) present in rat brain. Soluble KAT was purified from rat kidney and the amino acid sequences of four tryptic peptides determined. These peptides were found to belong to the amino acid sequence reported for rat kidney soluble cysteine conjugate β‐lyase, indicating that rat kidney KAT and β‐lyase represent the same molecular entity. Oligonucleotide probes derived from the β‐lyase cDNA were then used as primers for PCR of reverse‐transcribed rat brain poly(A)+RNA. After subcloning of the resulting PCR fragment and sequencing of the isolated rat brain clone, its oligonucleotide sequence was found to be identical to that reported for the β‐lyase cDNA. Further evidence that the isolated rat brain clone encoded for KAT was obtained by transfecting HEK‐293 cells with a construct containing the coding sequence for the enzyme. The transfected cells exhibited KAT activity and, in the presence of 2 mMpyruvate and 2‐oxoglutarate, theKmvalues forl‐kynurenine were 1.2 mMand 86.3 µM, respectively. Northern blot analysis of rat kidney, liver, and brain RNA revealed a single species of KAT/β

ISSN:0022-3042    

DOI:10.1046/j.1471-4159.1995.64041448.x

出版商:Blackwell Science Ltd    

年代:1995

数据来源: WILEY

摘要:

Abstract:We describe the use of a baculovirus expression system to overproduce human Cu,Zn‐superoxide dismutase (SOD).Spodoptera frugiperda(Sf21) insect cells infected with a baculovirus carrying the Cu,Zn‐SOD cDNA synthesized a large amount of Cu,Zn‐SOD apoprotein in the conventional medium. The SOD activity of the apoprotein, which was initially very low, increased in a dose‐dependent manner when Cu2+and Zn2+were added to the medium. Cells grown in media supplemented with Cu2+alone exhibited nearly maximal SOD activity. SOD activity reached 40% of the maximal level within 2 h after addition of Cu2+to postinfected cells cultivated for 3 days in the conventional medium, and the activity gradually increased thereafter. The protein produced by the infected cells was purified by a simple procedure involving two chromatographic steps, DE52 ion exchange and ACA54 gel filtration. Identification of the recombinant Cu,Zn‐SOD with the human erythrocyte enzyme was confirmed by immunochemical reactivity to anti‐human Cu,Zn‐SOD antibody and by partial amino acid sequencing of peptides from purified protein (50 amino acid residues in total). We constructed three mutant enzymes, which have been found in familial amyotrophic lateral sclerosis and are overproduced in Sf21 cells, and purified them. Mutant enzymes Gly41Asp, His43Arg, and Gly85Arg exhibited 47, 66, and 99% of wild‐type SOD activity, respectively. The availability of this protein will facilitate investigation of the relationship between the structure and function of the mutant enzymes found in familial amyotrophic la

ISSN:0022-3042    

DOI:10.1046/j.1471-4159.1995.64041456.x

出版商:Blackwell Science Ltd    

年代:1995

数据来源: WILEY

摘要:

Abstract:Pharmacological and molecular biological evidence indicates the existence of multiple types of NMDA receptors within the CNS. We have characterized pharmacological properties of receptors assembled from the combination of NR 1a and NR 2B subunits (NR 1a/2B) expressed in transfected cells using both125I‐MK‐801 binding assays and electrophysiological measures. Binding of125I‐MK‐801 to cells transfected with NR 1a/2B is saturable with aKDof 440 pM. The binding is potently inhibited by ketamine, dextromethorphan, phencyclidine, and MK‐801 and is stimulated by low concentrations of magnesium. These properties resemble those of native receptors and receptors produced by NR 1a/2A. However,125I‐MK‐801 binding to membranes from cells transfected with NR 1a/2B is inhibited with high affinity by ifenprodil and is stimulated by spermidine, unlike receptors assembled from NR 1a/2A. NMDA‐induced currents measured in cells transfected with either NR 1a/2A or NR 1a/2B have pharmacological properties that correlate well with the binding studies. Currents in cells transfected with NR 1a/2B are potentiated by spermidine and blocked with high affinity by ifenprodil, whereas currents in cells transfected with NR 1a/2A are not enhanced by spermidine and are weakly inhibited by ifenprodil. These data suggest that pharmacological heterogeneity in native NMDA receptors may be explained by combinations of dif

ISSN:0022-3042    

DOI:10.1046/j.1471-4159.1995.64041462.x

出版商:Blackwell Science Ltd    

年代:1995

数据来源: WILEY

摘要:

Abstract:In the present study we investigated uptake of the nitric oxide (NO) synthase inhibitorsNG‐methyl‐l‐arginine andNG‐nitro‐l‐arginine by the mouse neuroblastoma × rat glioma hybrid cell line NG108‐15. Uptake ofNG‐methyl‐l‐arginine was characterized by biphasic kinetics (Km1= 8 µmol/L,Vmax1= 0.09 nmol × mg−1× min−1;Km2= 229 µmol/L,Vmax2= 2.9 nmol × mg−1× min−1) and was inhibited by basic but not by neutral amino acids. Uptake ofNG‐nitro‐l‐arginine followed Michaelis‐Menten kinetics (Km= 265 µmol/L,Vmax= 12.8 ± 0.86 nmol × mg−1× min−1) and was selectively inhibited by aromatic and branched chain amino acids. Further characterization of the transport systems revealed that uptake ofNG‐methyl‐l‐arginine is mediated by system y+, whereas systems L and T account for the transport ofNG‐nitro‐l‐arginine. In agreement with these data on uptake of the inhibitors,l‐lysine andl‐ornithine antagonized the inhibitory effects ofNG‐methyl‐l‐arginine on bradykinin‐induced intracellular cyclic GMP accumulation, whereasl‐tryptophan,l‐phenylalanine, andl‐leucine interfered with the effects ofNG‐nitro‐l‐arginine. These data suggest

ISSN:0022-3042    

DOI:10.1046/j.1471-4159.1995.64041469.x

出版商:Blackwell Science Ltd    

年代:1995

数据来源: WILEY

摘要:

Abstract:Interferon (IFN)‐β and IFN‐γ inhibited the DNA synthesis and nerve growth factor (NGF) synthesis in growing astrocytes cultured from neonatal mouse brain, but they did not affect the NGF synthesis in quiescent astrocytes. IFN‐β and IFN‐γ also inhibited the enhanced DNA synthesis and NGF synthesis in growing astrocytes after the administration of basic fibroblast growth factor. These results indicated that NGF synthesis in astrocytes is regulated by IFNs associated with cell growth. The mechanism of IFN action on NGF synthesis/secretion is unknown, but the results that their effects last long after IFN removal from the cultures present the possibility that IFNs destabili

ISSN:0022-3042    

DOI:10.1046/j.1471-4159.1995.64041476.x

出版商:Blackwell Science Ltd    

年代:1995

数据来源: WILEY

摘要:

Abstract:In the human neuroblastoma cell line LA‐N‐2, recombinant rat ciliary neurotrophic factor (CNTF) induced neurite growth and cholinergic differentiation that were both half‐maximally saturated at<100 pMof the neurokine, but was not required for cell survival in serum‐free conditions over a 13‐day period. CNTF markedly stimulated choline acetyltransferase activity and acetylcholine synthesis, whereas high‐affinity choline transport was only slightly enhanced and acetylcholinesterase activity was unchanged. Leukemia inhibitory factor had effects identical to CNTF on neurite growth and choline acetyltransferase activity, but interleukin 6 had no effect. Radioiodinated CNTF binding and affinity cross‐linking studies were consistent with tripartite receptor activation as a mediator of the observed biolo

ISSN:0022-3042    

DOI:10.1046/j.1471-4159.1995.64041483.x

出版商:Blackwell Science Ltd    

年代:1995

数据来源: WILEY

摘要:

Abstract:Using video‐enhanced microscopy and a pulse‐radiolabeling paradigm, we show that proteins synthesized in the medial giant axon cell body of the crayfish (Procambarus clarkii) are delivered to the axon via fast (∼62 mm/day) and slow (∼0.8 mm/day) transport components. These data confirm that the medial giant axon cell body provides protein to the axon in a manner similar to that reported for mammalian axons. Unlike mammalian axons, the distal (anucleate) portion of a medial giant axon remains intact and functional for>7 months after severance. This axonal viability persists long after fast transport has ceased and after the slow wave front of radiolabeled protein has reached the terminals. These data are consistent with the hypothesis that another source (i.e., local glial cells) provides a significant amount of protein to supplement that delivered to the medial giant axon by its ce

ISSN:0022-3042    

DOI:10.1046/j.1471-4159.1995.64041491.x

出版商:Blackwell Science Ltd    

年代:1995

数据来源: WILEY

摘要:

Abstract:The organic molecule K‐252a promoted cell survival, neurite outgrowth, and increased choline acetyltransferase (ChAT) activity in rat embryonic striatal and basal forebrain cultures in a concentration‐dependent manner. A two‐ to threefold increase in survival was observed at 75 nMK‐252a in both systems. A single application of K‐252a at culture initiation prevented substantial (>60%) cell death that otherwise occurred after 4 days in striatal or basal forebrain cultures. A 5‐h exposure of striatal or basal forebrain cells to K‐252a, followed by its removal, resulted in survival equivalent to that observed in cultures continually maintained in its presence. This is in contrast to results found with a 5‐h exposure of basal forebrain cultures to nerve growth factor (NGF). Acute exposure of basal forebrain cultures to K‐252a, but not to NGF, increased ChAT activity, indicating that NGF was required the entire culture period for maximum activity. Striatal cholinergic and GABAergic neurons were among the neurons rescued by K‐252a. Of the protein growth factors tested in striatal cultures (ciliary neurotrophic factor, neurotrophin‐3, NGF, brain‐derived neurotrophic factor, interleukin‐2, basic fibroblast growth factor), only brain‐derived neurotrophic factor promoted survival. The enhancement of survival and ChAT activity of basal forebrain and striatal neurons by K‐252a defines additional populations of neurons in which survival and/or differentiation is regulated by a K‐252a‐responsive mechanism. The above results expand the potential therapeutic targets for these molecules for the treatm

ISSN:0022-3042    

DOI:10.1046/j.1471-4159.1995.64041502.x

出版商:Blackwell Science Ltd    

年代:1995

数据来源: WILEY