ISSN:0022-3042    

DOI:10.1046/j.1471-4159.1996.67030889.x

出版商:Blackwell Science Ltd    

年代:1996

数据来源: WILEY

摘要:

Abstract:A cDNA encoding full‐length tryptophan hydroxylase was produced by reverse transcriptase‐PCR from rat brain mRNA and expressed transiently in a human fibroblast cell line. Catalytic activity was low unless transfected cells were grown in the presence of FeSO4. Recombinant tryptophan hydroxylase was found almost exclusively within the soluble compartment of the cell and was dependent on tryptophan and tetrahydrobiopterin for activity. The catalytic activity of recombinant tryptophan hydroxylase was stimulated>25‐fold by Fe(II) and to a somewhat lesser extent by the polyanions heparin and phosphatidylserine. The enzyme was inhibited by desferrioxamine and dopamine, both of which complex iron. When extracts from transfected cells were subjected to sucrose gradient centrifugation and analytical gel filtration, the recombinant enzyme behaved the same as the native enzyme from brain. A monoclonal antibody against phenylalanine hydroxylase that cross‐reacts with brain tryptophan hydroxylase was capable of immunoprecipitating the recombinant hydroxylase from solution. These data indicate that recombinant tryptophan hydroxylase expressed in mammalian cells is assembled into tetramers of ∼220,000 daltons. Its catalytic and physical properties appear to be very similar to those of the native enzyme f

ISSN:0022-3042    

DOI:10.1046/j.1471-4159.1996.67030900.x

出版商:Blackwell Science Ltd    

年代:1996

数据来源: WILEY

摘要:

Abstract:The α6 subunit of γ‐aminobutyric type A receptors is a marker for cerebellar granule cells and is an attractive candidate to study cell‐specific gene expression in the brain. The mouse α6 subunit gene has nine exons and spans ∼14 kb. The largest intron (intron 8) is ∼7 kb. For a minority of mRNAs, a missplice of the first exon was identified that disrupts the signal peptide and most likely results in the production of nonfunctional protein. The gene is transcribed from a TATA‐less promoter that uses multiple start sites. Using transgenic mice, it was found that the proximal 0.5 kb of the rat α6 gene upstream region confers expression on a β‐galactosidase reporter gene. One founder gave rise to a line with cerebellar granule cell‐specific expression, although expression varied with lobule region. Other founders had ectopic but neuron‐specific expression, with β‐galactosidase found in cerebellar Purkinje cells, neocortex, thalamus, hippocampus, caudate‐putamen, and inferior colliculi. Thus, we have defined a region containing the basal promoter of the α6 subunit gene and that confers

ISSN:0022-3042    

DOI:10.1046/j.1471-4159.1996.67030907.x

出版商:Blackwell Science Ltd    

年代:1996

数据来源: WILEY

摘要:

Abstract:cDNAs encoding the full‐length sequence for tryptophan hydroxylase, and deletion mutants consisting of the regulatory (amino acids 1–98) or catalytic (amino acids 99–444) domains of the enzyme, were cloned and expressed as glutathioneS‐transferase fusion proteins inE. coli. The recombinant fusion proteins could be purified to near homogeneity within minutes by affinity chromatography on glutathione‐agarose. The full‐length enzyme and the catalytic core expressed very high levels of tryptophan hydroxylase activity. The regulatory domain was devoid of activity. The full‐length enzyme and the catalytic core, while adsorbed to glutathione‐agarose beads, obeyed Michaelis‐Menten kinetics, and the kinetic properties of each recombinant enzyme for cofactor and substrate compared very closely to native, brain tryptophan hydroxylase. Both active forms of the glutathioneS‐transferase‐tryptophan hydroxylase fusion proteins had strict requirements for ferrous iron in catalysis and expressed much higher levels of activity (Vmax) than the brain enzyme. Analysis of full‐length tryptophan hydroxylase and the catalytic core by molecular sieve chromatography under nondenaturing conditions revealed that each fusion protein behaved as a tetrameric species. These results indicate that a truncated tryptophan hydroxylase, consisting of amino acids 99–444 of the full‐length enzyme, contains the sequence motifs needed for subunit assembly. Both wild‐type tryptophan hydroxylase and the catalytic core are expressed as apoenzymes which are converted to holoenzymes by exogenous iron. The tryptophan hydroxylase catalytic core is also as active as the full‐length enzyme, suggesting the possibility that the regulatory domain exerts a suppressive effect on the catalytic

ISSN:0022-3042    

DOI:10.1046/j.1471-4159.1996.67030917.x

出版商:Blackwell Science Ltd    

年代:1996

数据来源: WILEY

摘要:

Abstract:Dynamin proteins are members of a recently described family of GTPases involved in receptor‐mediated processes. To date, three different dynamin‐encoding genes have been identified in mammalian tissues. Dynamin I is expressed only in neurons, whereas dynamin II is ubiquitously expressed. A third isoform, dynamin III, was originally isolated from a rat testis cDNA library and shown to be testis‐specific. However, here we report the cloning and characterization of dynamin III from brain and lung, demonstrating a more extended pattern of expression for this isoform. In addition, we have investigated the temporal pattern of expression of these three genes during brain development. We find that both dynamin I and dynamin III mRNA levels are up‐regulated during embryogenesis, whereas dynamin II mRNA levels remain unchanged. From these results, we conclude that dynamin III is not a testis‐specific isoform and, furthermore, that rat brain expresses three different dynamin‐encoding genes that are differentially regulated during development. Therefore, this large isoform diversity of dynamin proteins in brain predicts a significant complexity in the understanding of dynamin‐based processes i

ISSN:0022-3042    

DOI:10.1046/j.1471-4159.1996.67030927.x

出版商:Blackwell Science Ltd    

年代:1996

数据来源: WILEY

摘要:

Abstract:We report here the sequences of two new proteins fromAplysia, aplycalcin andAplysianeurocalcin. These proteins belong to a family of calcium‐binding proteins, found primarily in vertebrate brain and retina, that have been proposed to play a role in calcium‐dependent regulation of enzymes in signal transduction pathways. Like other members of this family, theAplysiaproteins have consensus sequences for myristoylation, bind calcium, and translocate from cytosol to membrane when the calcium level is raised above the resting intracellular concentration. Both proteins are relatively enriched inAplysianervous system, but are also found to a significant degree in other tissues. The expression of mRNA for these proteins inAplysianervous tissue is regulated during development, roughly paralleling the reported emergence of several forms of synaptic plasticity. The messages are present at low levels in stage 11, show a large increase by late stage 12, and decline to a plateau of ∼30% of the peak value afterward. On the basis of the properties of these proteins and by analogy with proposed functions of some of the retinal homologues, we suggest that these proteins may play a role in mediating calcium‐dependent processes in neuronal function. The presence of both proteins in other tissues may suggest analogous roles for the proteins in other cel

ISSN:0022-3042    

DOI:10.1046/j.1471-4159.1996.67030932.x

出版商:Blackwell Science Ltd    

年代:1996

数据来源: WILEY

摘要:

Abstract:2′,3′‐Cyclic nucleotide 3′‐phosphodiesterase (CNP) is an isoprenylated protein enriched in myelin and oligodendrocytes but also present in several other tissues at low levels. CNP binds avidly to membranes and in addition possesses several characteristics of cytoskeletal proteins. The role of isoprenylation in the association of CNP with the cytoskeleton was analyzed by ectopic expression in L cells of epitope‐tagged CNP1 and a non‐isoprenylated mutant CNP1. Using nonionic detergent extraction, drug‐mediated cytoskeletal disruption, and coimmunoprecipitation with an anti‐actin antibody, we show that CNP1 is associated with actin‐based cytoskeletal elements independently of its isoprenylation status. A control protein, p21c‐H‐ras, which is also modified by isoprenylation at its carboxyl‐terminus, does not bind to cytoskeletal structures as judged by the same criteria. We present a model that accounts for the association of CNP1 with membr

ISSN:0022-3042    

DOI:10.1046/j.1471-4159.1996.67030943.x

出版商:Blackwell Science Ltd    

年代:1996

数据来源: WILEY

摘要:

Abstract:Expression of the neurotrophin‐3 (NT‐3) receptor (TrkC) and the effects of NT‐3 on signal transduction were investigated in highly enriched populations of embryonic rat hippocampal pyramidal neurons grown in bilaminar cultures. PCR analysis revealed that the predominanttrkCisoform is K1, which lacks an insert in the kinase domain. Polyclonal TrkC‐specific antibodies stained>90% of the neurons and revealed a single ∼145‐kDa protein in immunoblots of extracts from adult hippocampus and pyramidal neuron cultures. Addition of NT‐3 (50 mg/ml) to these cultures induced the tyrosine phosphorylation of TrkC but not TrkB, as determined by anti‐phosphotyrosine staining of immunoprecipitates; thus, all the effects of NT‐3 are mediated through TrkC. NT‐3 also increased the tyrosine phosphorylation of 42‐, 44‐, 49‐, 55‐, 95‐, and 145‐kDa proteins; the pattern induced by brain‐derived neurotrophic factor (BDNF) was similar but not identical to that induced by NT‐3, suggesting that subtle differences may exist in signaling by TrkB and TrkC receptors. Immunoprecipitation of p21rasfrom32P‐prelabeled cells showed that NT‐3 increased the level of the GTP‐bound form of the protein threefold over the control within 5 min. Mitogen‐activated protein (MAP) kinase activity was maximally elevated by NT‐3 within 2 min and then returned slowly toward baseline over the next 60 min. Tyrosine phosphorylation of phospholipase C‐γ increased rapidly after NT‐3, suggesting that this enzyme becomes activated. Consistent with this, the neurotrophin rapidly increased protein kinase C activity as well as intracellular Ca2+levels. The effects of both NT‐3 and BDNF on Ca2+levels were attenuated in Ca2+‐free medium, suggesting that both neurotrophins increase Ca2+flux across the plasma membrane as well as release from internal stores. NT‐3 also increased c‐Fos expression in>80% of the cells; the effect peaked at 30 minand declined to baseline by 120 min. Despite the activation of ras‐MAP kinase and phosphoinositide signaling pathways, neither NT‐3 nor BDNF alone or in combination could sustain hippocampal pyramidal neurons deprived of glial support. We conclude that in this system NT‐3 and BDNF do not appear to be acting as classical “neurotrophic” factors and that activation of the MAP k

ISSN:0022-3042    

DOI:10.1046/j.1471-4159.1996.67030952.x

出版商:Blackwell Science Ltd    

年代:1996

数据来源: WILEY

摘要:

Abstract:To extrapolate the function of the leptomeninges, we examined the profile of the proteins secreted from the cultured leptomeningeal cells prepared from 1–2‐day‐old rats. In sodium dodecyl sulfate‐polyacrylamide gel electrophoresis analysis of the medium conditioned with the cultured cells, 20–25 differentially distinctive protein bands were noted. Through several chromatographic procedures (Sephadex G‐75, Mono Q, and 7C8‐300), altogether 18 proteins were purified to homogeneity, and the partial amino acid sequence of each protein was determined. Homology search revealed that the major proteins included prostaglandin‐d‐synthase or β‐trace protein, insulin‐like growth factor (IGF)‐II, IGF‐binding protein‐2, apolipoprotein E, β2‐microglobulin, cystatin C, transferrin, peptidyl‐prolylcis‐transisomerase or cyclophilin C, secreted protein acidic and rich in cysteine, ubiquitin, lysozyme C, extracellular superoxide dismutase, and collagen α‐1 (III). Most of these proteins are known to be the major brain‐derived protein constituents of CSF and are thought to play important roles in certain biological events in the brain. Considering the morphological features, the present findings suggest the importance of the leptomenin

ISSN:0022-3042    

DOI:10.1046/j.1471-4159.1996.67030964.x

出版商:Blackwell Science Ltd    

年代:1996

数据来源: WILEY

摘要:

Abstract:The kinetic characteristics of [3H]adenosine uptake, the extent to which accumulated [3H]adenosine was metabolized, the effects such metabolism had on measurements of apparent Michaelis‐Menten kinetic values ofKTandVmax, and the sensitivities with which nucleoside transport inhibitors blocked [3H]adenosine accumulations were determined in cultured human fetal astrocytes.KTandVmaxvalues for accumulations of [3H]‐labeled purines using 15‐s incubations in the absence of the adenosine deaminase inhibitorerythro‐9‐(2‐hydroxy‐3‐nonyl)adenine (EHNA) and the adenosine kinase inhibitor 5′‐iodotubercidin (ITU) were 6.2 µMand 0.15 nmol/min/mg of protein for the high‐affinity and 2.6 mMand 21 nmol/min/mg of protein for the low‐affinity components respectively. In the presence of EHNA and ITU, where<4% of accumulated [3H]adenosine was metabolized, transport per se was measured, and kinetic values forKTandVmaxwere 179 µMand 5.2 nmol/min/mg of protein, respectively. In the absence of EHNA and ITU, accumulated [3H]adenosine was rapidly metabolized to AMP, ADP, and ATP, and caused an appearance of “concentrative” uptake in that the intracellular levels of [3H]‐labeled purines (adenosine plus its metabolites) were 1.4‐fold higher than in the medium. No apparent concentrative accumulations of [3H]adenosine were found when assays were conducted using short incubation times in the absence or presence of EHNA and ITU. The nucleoside transport inhibitors dipyridamole (DPR), nitrobenzylthioinosine (NBI), and dilazep biphasically inhibited [3H]adenosine transport; for the inhibitor‐sensitive components the IC50values were 0.7 nMfor NBI, 1.3 nMfor DPR, and 3.3 nMfor dilazep, and for the inhibitor‐resistant component the IC50values were 2.5 µMfor NBI, 5.1 µMfor dilazep, and 39.0 µMfor DPR. These findings, in cultured human fetal astrocytes, represent the first demonstration of inhibitor‐sensitive and ‐resistant adenosin

ISSN:0022-3042    

DOI:10.1046/j.1471-4159.1996.67030972.x

出版商:Blackwell Science Ltd    

年代:1996

数据来源: WILEY