摘要:

Abstract:We describe the molecular cloning of a cDNA encoding a human brain Na+‐dependent inorganic phosphate (Pi) cotransporter (hBNPI). The nucleotide and deduced amino acid sequences of hBNPI reveal a protein of 560 amino acids with six to eight putative transmembrane segments. hBNPI shares a high degree of homology with other Na+‐dependent inorganic Picotransporters, including those found in rat brain and human and rabbit kidney. Expression of hBNPI in COS‐1 cells results in Na+‐dependent Piuptake. Northern blot analysis demonstrates that hBNPI mRNA is expressed predominantly in brain and most abundantly in neuron‐enriched regions such as the amygdala and hippocampus. Moderate levels of expression are also observed in glia‐enriched areas such as the corpus callosum, and low levels are observed in the substantia nigra, subthalamic nuclei, and thalamus. In situ hybridization histochemistry reveals relatively high levels of hBNPI mRNA in pyramidal neurons of the cerebral cortex and hippocampus and in granule neurons of dentate gyrus. The level of hBNPI mRNA is quite low in fetal compared with adult human brain, suggesting developmental regulation of hBNPI gene expression. Southern analyses of nine eukaryotic genomic DNAs probed under stringent conditions with hBNPI cDNA revealed that the hBNPI gene is highly conserved during vertebrate evolution and that each gene is most likely present as a single copy. Using fluorescent in situ hybridization, we localized hBNPI to the long arm of chromosome 19 (19q13) in close proximity to the late‐onset familial Alzheimer's

ISSN:0022-3042    

DOI:10.1046/j.1471-4159.1996.66062227.x

出版商:Blackwell Science Ltd    

年代:1996

数据来源: WILEY

摘要:

Abstract:Transfection of mouse L(tk‐) cells with humanN‐methyl‐d‐aspartate (NMDA) receptor subunit cDNAs under the control of a dexamethasone‐inducible promoter has been used to generate two stable cell lines expressing NR1a/NR2A receptors and a stable cell line expressing NR1a/NR2B receptors. The cell lines have been characterised by northern and western blot analyses, and the pharmacology of the recombinant receptors determined by radioligand binding techniques. Pharmacological differences were identified between the two NMDA receptor subtypes. The glutamate site antagonistd,l‐(ε)‐2‐[3H]amino‐4‐propyl‐5‐phosphono‐3‐pentanoic acid ([3H]CGP 39653) had high affinity for NR1a/NR2A receptors (KD= 3.93 nM) but did not bind to NR1a/NR2B receptors. Glycine site agonists showed a 2.6–5.4‐fold higher affinity for NR1a/NR2B receptors. Data from radioligand binding studies indicated that one of the cell lines, NR1a/NR2A‐I, expressed a stoichiometric excess of the NR1a subunit, which may exist as homomeric assemblies. This observation has implications when interpreting data from pharmacological analysis of recombinant receptors, as well as understanding the assembly and control of e

ISSN:0022-3042    

DOI:10.1046/j.1471-4159.1996.66062239.x

出版商:Blackwell Science Ltd    

年代:1996

数据来源: WILEY

摘要:

Abstract:Chromogranins and secretogranins are acidic secretory proteins of unknown function that represent major constituents of neuroendocrine secretory granules. Using a differential screening strategy designed to identify genes involved in peptide hormone biosynthesis and secretion, we have isolated cDNA clones encoding the first nonmammalian homologues of secretogranin II (SgII) and secretogranin III (SgIII) from aXenopusintermediate pituitary cDNA library. A comparative analysis of theXenopusand mammalian proteins revealed a striking regional conservation with an overall sequence identity of 48% for SgII and 61% for SgIII. One of the highly conserved and thus potentially functional domains in SgII corresponds to the bioactive peptide secretoneurin. However, in SgII and especially in SgIII, a substantial portion of the potential dibasic cleavage sites is not conserved, arguing against the idea that these granins serve solely as peptide precursors. Moreover, SgIII contains a conserved and repeated motif (DSTK) that is reminiscent of a repeat present in thetrans‐Golgi network integral membrane proteins TGN38 and TGN41, a finding more consistent with an intracellular function for this protein. WhenXenopusintermediate pituitary cells were stimulated in vivo, the mRNA levels of SgII and SgIII increased dramatically (15‐ and 35‐fold, respectively) and in parallel with that of the prohormone proopiomelanocortin (30‐fold increase). Our results indicate that the process of peptide hormone production and release in a neuroendocrine cell involves multiple members of the granin

ISSN:0022-3042    

DOI:10.1046/j.1471-4159.1996.66062248.x

出版商:Blackwell Science Ltd    

年代:1996

数据来源: WILEY

摘要:

Abstract:The microtubule‐associated protein τ is produced from a 6‐kb mRNA expressed primarily in neurons. A 2‐kb τ mRNA has also been characterized, which produces a τ isoform that localizes to the nucleus, and an 8‐kb mRNA is expressed in the PNS. Mapping and sequencing of the human τ gene start showed that it has an unusually GC‐rich 5′‐untranslated region coded by a single untranslated exon (designated −1). Primer extensions and expression assays indicated that upstream of exon −1 is a promoter that is not neuron specific. This region contains consensus binding sites for transcription fact

ISSN:0022-3042    

DOI:10.1046/j.1471-4159.1996.66062257.x

出版商:Blackwell Science Ltd    

年代:1996

数据来源: WILEY

摘要:

Abstract:In bovine chromaffin cells forskolin, phorbol ester, or high potassium levels induce a rapid increase of c‐fos, c‐jun, andjunBmRNA levels, which precede an induction of proenkephalin gene expression. Preincubation of the cells with cycloheximide inhibited induction of proenkephalin mRNA levels by each of these agents, indicating that newly synthesized transcription factors are involved. Transient transfection of reporter genes showed that the ENKCRE‐2 element of the proenkephalin promoter was sufficient for basal and second messenger‐induced expression. Gel mobility shift assays revealed that stimulation increased the binding of nuclear proteins to ENKCRE‐2 and AP‐1 oligonucleotides but not to CRE oligonucleotides. Western analysis showed that the induction of AP‐1 binding activity was associated with Fos protein synthesis. Moreover, cotransfection of c‐fos, but not of c‐junor CREB, expression plasmids transactivated the expression of the PENKCAT reporter genes. These results suggest that Fos and/or other components of AP‐1 transcription factors, rather than CREB or other preexisting proteins, play a specific role in the induction of the proenkephalin gene in bovi

ISSN:0022-3042    

DOI:10.1046/j.1471-4159.1996.66062264.x

出版商:Blackwell Science Ltd    

年代:1996

数据来源: WILEY

摘要:

Abstract:The actions of exogenous and endogenous opioids are mediated by at least three different opioid receptors, called μ, κ, and δ. Recently, we have detected a new variant of the rat μ‐opioid receptor, which we termed rMOR1B and which differs from rMOR1 (now also called rMOR1A) in the amino acid sequence at the C‐terminus. Both isoforms were proposed to be splicing variants of the same gene. To elucidate the molecular mechanism leading to the formation of the new variant, the exon/intron structure of the rat μ‐opioid receptor gene in the respective area has been determined by analyzing a genomic P1 phage clone. In addition, we have investigated the putative promoter region of this gene. The present study revealed that rMOR1B is generated by an alternative splicing event whereby a previously unknown exon will be placed behind exon 3 to form rMOR1B mRNA, which is separated from the latter by an intron. Therefore, this new exon has to be called exon 4, whereas the former exon 4, which encodes the C‐terminus of MOR1A, now becomes exon 5. Examination of the putative rat promoter region revealed a high degree of nucleotide sequence homology to the mouse gene. Using an RNase protection approach, one single transcription initiation site could be located at 230 bp upstream of the translation start. This is similar to the situation in the mouse, where four major transcription start sites were reported to lie close together around 270 bp upstream of the protein c

ISSN:0022-3042    

DOI:10.1046/j.1471-4159.1996.66062272.x

出版商:Blackwell Science Ltd    

年代:1996

数据来源: WILEY

摘要:

Abstract:Brain‐derived neurotrophic factor (BDNF), a member of the neurotrophin family, regulates survival and apoptosis of several neuronal populations. These effects are initiated by high‐affinity membrane receptors displaying tyrosine kinase activity (trk). However, the intracellular pathways and genetic mechanisms associated with these receptors are largely unknown. Here we show that BDNF stimulates AP1 binding activity in primary cerebellar neurons. This binding corresponds to a functional complex as it is associated with the induction of AP1‐dependent transactivation. Application of AP1 partner mRNAs shows an increase in levels of c‐fosand c‐junmRNAs after BDNF treatment, resulting from an induction of their promoters. Thecis‐acting elements by which BDNF stimulates c‐fostranscription were further studied. We show that BDNF impinges on multiple regulatory elements, including the serum‐responsive element, Fos AP1‐like element, and cyclic AMP (cAMP)‐responsive element (CRE) sequences. The latter was stimulated without any detectable increase in cAMP or Ca2+levels. To confirm that BDNF induces c‐fostranscription independently of the protein kinase A/cAMP pathway, we transfected a dominant inhibitory mutant of the regulatory subunit of protein kinase A. The overexpression of this mutant does not affect the c‐fospromoter transactivation by BDNF. In summary, we show that BDNF stimulates AP1‐ and CRE‐dependent transcription through a mechanism that is distinct from the cAMP‐ and Ca2+‐de

ISSN:0022-3042    

DOI:10.1046/j.1471-4159.1996.66062279.x

出版商:Blackwell Science Ltd    

年代:1996

数据来源: WILEY

摘要:

Abstract:To investigate the role of the retinoblastoma protein pRB in neuronal differentiation, we have measured the accumulation of hypophosphorylated pRB in PC12 cells stimulated by nerve growth factor (NGF). NGF induced the accumulation of hypophosphorylated pRB within 30 min and the level peaked after 12 h. Viral Kiras, cyclic AMP (cAMP), and 12‐O‐tetradecanoylphorbol 13‐acetate (TPA) also induced the hypophosphorylation of pRB, but epidermal growth factor and interleukin‐6 did not. The extent of hypophosphorylation of pRB correlated well with the capacity of these factors to stimulate neurite outgrowth. The constitutively activated Ras induced persistent shift of the phosphorylation state of pRB toward hypophosphorylation. A dominant negative form of cHa‐Ras suppressed significantly induction of the hypophosphorylation of pRB by NGF, but not by cAMP. Taken together, these results suggest that the hypophosphorylation of pRB triggered by NGF is mediated by a Ras‐dependent pathway. Furthermore, microinjection of a monoclonal antibody specific for the hypophosphorylated form of pRB blocked the neurite outgrowth initiated by NGF. These results suggest a crucial role of pRB in withdrawal of cells from the cell cycle and in neuronal differentiation of

ISSN:0022-3042    

DOI:10.1046/j.1471-4159.1996.66062287.x

出版商:Blackwell Science Ltd    

年代:1996

数据来源: WILEY

摘要:

Abstract:In the present study we applied an improved nerve growth factor (NGF) extraction method to examine the effects of denervation and sympathetic decentralization on NGF levels in vascular tissue. Adult male Wistar Kyoto rats underwent mesenteric arterial denervation or splanchnic nerve transection. Four days after operation, animals were killed, and the mesenteric artery and coeliac‐superior mesenteric ganglia were removed. The arterial adventitia was stripped from the media to measure NGF levels in nerve and smooth muscle separately. A high concentration of NGF was detected in the normal artery, 90% of which was in the adventitial layer. Surgical denervation significantly reduced the NGF levels in the artery and ganglia by 78 and 71%, respectively. However, within the artery the level of NGF was reduced in the adventitia but not in the media. Thus, the large reduction of NGF content resulted from the loss of nerve plexus from the artery. In contrast, decentralization did not alter the NGF content in the artery, in either the adventitia or media. Our results are in marked contrast to previous studies reporting elevated levels of NGF following denervation. This discrepancy is explained by the ability of our new procedure to extract much greater amounts of NGF from the tissu

ISSN:0022-3042    

DOI:10.1046/j.1471-4159.1996.66062295.x

出版商:Blackwell Science Ltd    

年代:1996

数据来源: WILEY

摘要:

Abstract:Amyloid precursor protein (APP) gives rise by proteolytic processing to the amyloid β peptide (Aβ) found abundantly in cerebral senile plaques of individuals with Alzheimer's disease. APP is highly expressed in the brain. To assess the source of cerebral Aβ, the metabolism of APP was investigated in the major cell types of the newborn rat cerebral cortex by pulse/chase labeling and immunoprecipitation of the APP and APP metabolic fragments. We describe a novel C‐terminally truncated APP isoform that appears to be made only in neurons. The synthesis, degradation, and metabolism of APP were quantified by phosphorimaging in neurons, astrocytes, and microglia. The results show that although little APP is metabolized through the amyloidogenic pathways in each of the three cultures, neurons appear to generate more Aβ than astrocytes or micr

ISSN:0022-3042    

DOI:10.1046/j.1471-4159.1996.66062300.x

出版商:Blackwell Science Ltd    

年代:1996

数据来源: WILEY