摘要:

Abstract:Peptides are of potential interest in the field of gene therapy but require modification by genetic engineering to facilitate their secretion. Amino terminal addition of a signal peptide is not always sufficient to achieve this goal, as found in this study for β‐endorphin. To overcome this problem, addition of the pre‐pro‐sequence of mouse nerve growth factor to β‐endorphin was tested. Retrovirus‐mediated expression of a hybrid construct of the pre‐pro‐sequence of nerve growth factor and human β‐endorphin in primary fibroblasts resulted in the secretion of β‐endorphin immunoreactivity at a rate of 620 pg/h/106cells. Analysis of the secreted β‐endorphin immunoreactivity with reverse‐phase HPLC, immunoassays using three different antibodies, and an assay for the specific displacement of [3H][d‐Ala2,N‐MePhe4,Gly‐ol5]enkephalin from μ‐opioid receptors suggests that the pre‐pro‐sequence is cleaved off from the pre‐pro‐sequence/β‐endorphin construct prior to secretion, resulting in bona fide β‐endorphin. Transplantation of β‐endorphin‐secreting cells into brain or spinal cord may provide a gene therapy approach for

ISSN:0022-3042    

DOI:10.1046/j.1471-4159.1995.64020475.x

出版商:Blackwell Science Ltd    

年代:1995

数据来源: WILEY

摘要:

Abstract:We have cloned the gene encoding the murine D3dopamine receptor and have analyzed its intron‐exon structural organization, to gain a better understanding of the detailed architecture of the D2dopamine receptor genes. Restriction and sequence analysis reveal the presence of six introns, in contrast to the five introns previously reported for the rat D3receptor. The extra intron is located in the receptor's putative third cytoplasmic loop and generates an intron‐exon organization directly analogous to that found in the D2receptor gene. In addition, we have sequenced the 5′ and 3′ nontranslated sequences flanking the coding region and have identified a putative poly(A) adenylation signal. These sequences are found to have a far lower homology with the corresponding rat nontranslated sequences than is found for the D2receptor, suggesting that the control of D3receptor expression may vary more between species than the control of D2receptor exp

ISSN:0022-3042    

DOI:10.1046/j.1471-4159.1995.64020482.x

出版商:Blackwell Science Ltd    

年代:1995

数据来源: WILEY

摘要:

Abstract:In this report we demonstrate that a defective herpes simplex virus type one (HSV‐1) vector can express enzymatically active tyrosine hydroxylase in cultured striatal cells that are thereby converted intol‐DOPA‐producing cells. A human tyrosine hydroxylase cDNA (form II) was inserted into an HSV‐1 vector (pHSVth) and packaged into virus particles using an HSV‐1 strain 17 mutant in the immediate early 3 gene (either ts K or D30EBA) as helper virus. Cultured fibroblasts were infected with pHSVth and 1 day later tyrosine hydroxylase immunoreactivity and tyrosine hydroxylase enzyme activity were observed. The tyrosine hydroxylase enzyme activity directed the production ofl‐DOPA. pHSVth infection of striatal cells in dissociated cell culture resulted in expression of tyrosine hydroxylase RNA and tyrosine hydroxylase immunoreactivity. Release ofl‐DOPA and low levels of dopamine were observed from cells in pHSVth‐infected striatal cultures. Expression of tyrosine hydroxylase and release of catecholamines were maintained for at least 1 week

ISSN:0022-3042    

DOI:10.1046/j.1471-4159.1995.64020487.x

出版商:Blackwell Science Ltd    

年代:1995

数据来源: WILEY

摘要:

Abstract:A subtractive hybridization and differential screening procedure was used to detect up‐regulation of cytochromecoxidase (CO) subunits I, III, and IV mRNA in the nucleus accumbens (NAc) of rats chronically treated with cocaine. Northern blot analyses of mRNA isolated from individual rats confirmed that CO subunit I was up‐regulated by chronic, but not acute, cocaine in two brain regions, the NAc (33%) and caudate‐putamen (CP)(35%). CO activity, used as a measure of metabolic activity, was increased by 88% in the NAc, and decreased by 20% in the medial prefrontal cortex (mPFC), the day after chronic treatment was terminated. CO enzyme activity was not regulated in the CP, or in other brain regions not involved in drug reward. CO activity in both the NAc and mPFC showed unique time‐dependent patterns of regulation during the week after chronic cocaine tr

ISSN:0022-3042    

DOI:10.1046/j.1471-4159.1995.64020497.x

出版商:Blackwell Science Ltd    

年代:1995

数据来源: WILEY

摘要:

Abstract:Basic fibroblast growth factor (FGF‐2) is normally expressed as a cell‐associated protein, and accordingly it is not clear how it exerts its action on target cells in vivo. It has been proposed that cells release, by death or other mechanisms, small amounts of FGF‐2 that then acts in an autocrine manner. To address the question of whether it is necessary that FGF‐2 remain cell associated or needs to be secreted from cells to have biological activity, we expressed the 18‐kDa form of FGF‐2 in primary fibroblasts as a cell‐associated (FGF‐2‐B) or as a secreted (FGF‐2‐S) protein. FGF‐2 protein is detected in cell lysates and membrane fractions of both cell types, whereas it is present in significant amounts only in the conditioned medium of FGF‐2‐S cells. No FGF‐2 is detected in control (untransfected) cells. FGF‐2‐S cells also grow faster than the control or FGF‐2‐B cells. Yet, when evaluated for their ability to promote the survival of embryonic hippocampal neurons in vitro, both the cell types are active, establishing the activity of the transgene product. We conclude that FGF‐2 is active when engineered to be expressed as a cell

ISSN:0022-3042    

DOI:10.1046/j.1471-4159.1995.64020503.x

出版商:Blackwell Science Ltd    

年代:1995

数据来源: WILEY

摘要:

Abstract:Previously we identified two alternative first exons (exon N1 and exon L1) coding for 5′ untranslated regions of human aromaticl‐amino acid decarboxylase (AADC) and found that their alternative usage produced two types of mRNAs in a tissue‐specific manner. To determine thecis‐acting element regulating the tissue‐specific expression of human AADC, we produced three kinds of transgenic mice harboring 5′ flanking regions of the human AADC gene fused to the bacterial chloramphenicol acetyltransferase (CAT) gene. The transgene termed ACA contained −7.0 kb to −30 bp in exon N1, including the entire exon L1; ACN contained −3.6 kb to −30 bp in exon N1; and ACL contained −2.8 kb to −42 bp in exon L1. The ACA transgenic mice expressed CAT at extremely high levels in peripheral nonneuronal tissues, such as pancreas, liver, kidney, small intestine, and colon, that contained endogenous high AADC activity, whereas CAT immunoreactivity was not detected in either catecholaminergic or serotonergic neurons in the CNS. Thus, it was suggested that the ACA transgene contained the major part ofcis‐regulatory elements for the expression of AADC in peripheral nonneuronal tissues. On the other hand, the ACN transgenic mice moderately expressed CAT in various tissues except for the lung and liver, and the ACL transgenic mice showed moderate CAT expr

ISSN:0022-3042    

DOI:10.1046/j.1471-4159.1995.64020514.x

出版商:Blackwell Science Ltd    

年代:1995

数据来源: WILEY

摘要:

Abstract:The cDNA NMDAR1 (NR1) encodes a single polypeptide that forms a receptor‐channel complex with electrophysiological and pharmacological properties characteristic of theN‐methyl‐d‐aspartate receptor. Homomeric NR1 recombinant receptors expressed inXenopusoocytes show functional responses with low levels of conductance. In this study we have characterized, by radioligand binding techniques, the pharmacological properties of homomeric receptors of two human NR1 isoforms (NR1a and NR1e, which differ in their C‐terminal region), transiently expressed in human embryonic kidney 293 cells. The glycine site antagonist (±)‐4‐(trans)‐2‐carboxy‐5,7‐dichloro‐4‐[3H]phenylaminocarbonylamino‐1,2,3,4‐tetrahydroquinoline ([3H]L‐689,560) bound to NR1a‐ and NR1e‐transfected cells with high affinity (KD= 3.29 and 1.61 nM, respectively).Bmaxvalues for NR1a‐ and NR1e‐transfected cells were 3.82 and 1.69 pmol/mg of protein, respectively, and Hill coefficients were close to unity.Kivalues for glycine site antagonists inhibiting [3H]L‐689,560 binding to NR1e‐transfected cells were similar to those observed with rat brain membranes. Affinity values for agonists and partial agonists were four‐ to 16‐fold weaker, indicating that the glycine site of homomeric NR1 receptors is in an antagonist‐preferring state.Kivalues obtained with NR1a‐transfected cells were approximately twofold lower than those obtained with NR1e‐transfected cells. High‐affinity binding to NR1‐transfected cells was not observed with the transmitter recognition site radioligandsl‐[3H]glutamate andd,l‐(ε)‐2‐[3H]amino‐4‐propyl‐5‐phosphono‐3‐pentanoic acid ([3H]CGP‐39653) or the ion‐channel radioligand [3H]dizocilpine ([3H]MK‐801). These results indicate that although transfection of mammalian cells with homomeric NR1 recombinant receptors does not appear to result in functional receptors, a

ISSN:0022-3042    

DOI:10.1046/j.1471-4159.1995.64020525.x

出版商:Blackwell Science Ltd    

年代:1995

数据来源: WILEY

摘要:

Abstract:Developmental changes in the levels ofN‐methyl‐d‐aspartate (NMDA) receptor subunit mRNAs were identified in rat brain using solution hybridization/RNase protection assays. Pronounced increases in the levels of mRNAs encoding NR1 and NR2A were seen in the cerebral cortex, hippocampus, and cerebellum between postnatal days 7 and 20. In cortex and hippocampus, the expression of NR2B mRNA was high in neonatal rats and remained relatively constant over time. In contrast, in cerebellum, the level of NR2B mRNA was highest at postnatal day 1 and declined to undetectable levels by postnatal day 28. NR2C mRNA was not detectable in cerebellum before postnatal day 11, after which it increased to reach adult levels by postnatal day 28. In cortex, the expression of NR2A and NR2B mRNAs corresponds to the previously described developmental profile of NMDA receptor subtypes having low and high affinities for ifenprodil, i.e., a delayed expression of NR2A correlating with the late expression of low‐affinity ifenprodil sites. In cortex and hippocampus, the predominant splice variants of NR1 were those without the 5′ insert and with or without both 3′ inserts. In cerebellum, however, the major NR1 variants were those containing the 5′ insert and lacking both 3′ inserts. The results show that the expression of NR1 splice variants and NR2 subunits is differentially regulated in various brain regions during development. Changes in subunit expression are likely to underlie some of the changes in the functional and pharmacological properties of NMDA receptors that occur dur

ISSN:0022-3042    

DOI:10.1046/j.1471-4159.1995.64020531.x

出版商:Blackwell Science Ltd    

年代:1995

数据来源: WILEY

摘要:

Abstract:The protein kinase inhibitor K‐252a has been shown to promote cholinergic activity in cultures of rat spinal cord and neuronal survival in chick dorsal root ganglion cultures. To determine the mechanism by which K‐252a acts as a neurotrophic factor, we examined the effects of this molecule on a human neuroblastoma cell line, SH‐SY5Y. K‐252a induced neurite outgrowth in a dose‐dependent manner. Coincident with neurite outgrowth was the early tyrosine phosphorylation of 125‐ and 140‐kDa proteins. The phosphorylation events were independent of protein kinase C inhibition because down‐regulation of protein kinase C by long‐term treatment with phorbol ester did not prevent K‐252a‐induced tyrosine phosphorylation. Similarly, the protein kinase C inhibitors H7, GF‐109203X, and calphostin C did not induce the phosphorylation. We have identified one of the phosphosubstrates as the pp125 focal adhesion protein tyrosine kinase (Fak). Induction of phosphorylation coincided with increased Fak activity and appeared to be independent of ligand/integrin interaction. The induction of Fak phosphorylation by K‐252a was also observed in LA‐N‐5 cells and primary cultures of rat embryonic striatal cells but not in PC12 cells. The protein kinase C‐independent induction of tyrosine phosphorylation and the identification of Fak as a substrate of K‐252a‐induced tyrosine kinase activity suggest that this compound mediates neurotrophic effect

ISSN:0022-3042    

DOI:10.1046/j.1471-4159.1995.64020540.x

出版商:Blackwell Science Ltd    

年代:1995

数据来源: WILEY

摘要:

Abstract:When PC12 cells are primed with nerve growth factor (NGF) for periods of ≥1 week, they acquire the ability to regenerate neurites rapidly in response to NGF. It is not known how NGF promotes this regeneration, but it does not require ongoing RNA synthesis. Previous studies have suggested that NGF directs the accumulation of precursor molecules that are rapidly assembled to form the regenerated neurites. To address the nature of these precursor molecules, we have treated PC12 cells with macromolecular synthesis inhibitors during the priming and regeneration phases of neurite growth. Here we show that NGF promotes neurite regeneration by inducing the synthesis of new proteins. These proteins are encoded by short‐lived mRNAs that are generated during the NGF priming period. The isolation and identification of these mRNAs will allow a further understanding of how NGF promotes neurite regenerat

ISSN:0022-3042    

DOI:10.1046/j.1471-4159.1995.64020550.x

出版商:Blackwell Science Ltd    

年代:1995

数据来源: WILEY