Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:Q9NRP7 (fused)
 58,367 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We cloned, sequenced and characterized a promoter region of the mouse homologue of the Alzheimer's disease amyloid precursor protein (APP)-encoding gene. The promoter region is highly homologous to that of the human APP (hAPP) gene. It has a high G+C content, lacks typical 'TATA' and 'CAAT' boxes, and contains possible binding sites for AP-1, heat-shock factor, Sp1 and AP-4. The promoter region was fused with the cat reporter gene, and the fusion genes were transfected to both the NB41A3 (mouse neuroblastoma) and L-cell lines. The promoter activity was monitored by chloramphenicol acetyltransferase (CAT) activity in a transient expression assay. The promoter was equally active in both cell lines. The deletion analysis revealed that there existed a negative regulatory element(s) between -153 and -100 bp and a positive element(s) between -100 and -37 bp. The negative element was shown to suppress the transcriptional activity of heterologous simian virus 40 promoter. DNase I footprinting experiments revealed that three nuclear protein-binding sites existed in the regulatory region, one in the negative and two in the positive regulatory regions. Gel retardation assay showed that Sp1 was one of the factors binding to the positive regulatory region. A nuclear factor binding to the negative regulatory region seemed to be missing in brain.
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PMID:Positive and negative regulatory elements for the expression of the Alzheimer's disease amyloid precursor-encoding gene in mouse. 155 68

Transcriptional regulation of the gene encoding the amyloid precursor protein (APP) may play an important role in the formation of the amyloid depositions observed in Alzheimer disease and Down syndrome patients. To determine the promoter sequence requirements for the expression of the APP gene, we constructed plasmids containing different parts of the APP gene promoter fused to the bacterial chloramphenicol acetyl transferase (CAT) gene. Transfection of these constructs into Hela and PC12 cells revealed the presence of two blocks of regulatory sequences in the APP promoter. One block extending from about -600 to -460 bp acts as a positive regulator as its removal results in a substantial decrease in promoter activity. A second block of sequences extending from -450 to -150 bp acts like a negative regulator. We have also observed that a 38 mer synthetic oligonucleotide encompassing the region -489 to -452 of the APP promoter stimulated the activity of the heterologous TK promoter, suggesting that this region may be part of an enhancer-like element. In addition, our results suggest that the effects of various APP promoter domains on its activity may be cell specific.
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PMID:The promoter activity of the gene encoding Alzheimer beta-amyloid precursor protein (APP) is regulated by two blocks of upstream sequences. 185 27

A 3.3-kilobase DNA complementary to human microtubule-associated protein 2 (MAP2) was sequenced by the dideoxy method. The 3' end terminates at an internal EcoRI site before the polyA tail. Due to the arrangement of the cDNA insert in the lambda gt11 vector, the MAP2 fragment is not fused to beta-galactosidase when expressed. The Chou Fasman algorithm for the initial 58 amino acids from the first in-frame methionine predicts an alpha helix. Beyond this point, a series of turns is predicted until amino acid 160. The frequent presence of basic residues in proximity to serines or threonines is consistent with multiple phosphorylation sites. The minimum specificity determinant for Ca2+/calmodulin-dependent kinase is repeated 13 times. The sequence of a region containing a MAP2 epitope that is shared with the Alzheimer neurofibrillary tangle was determined by DNase treatment of the cDNA and antibody selecting the small resultant clones in a lambda gt11 sublibrary. Likewise, a MAP2 epitope that is not shared with the neurofibrillary tangle also has been located. Both epitopes are in the projection portion of the molecule. A bovine MAP2 cyanogen bromide fragment, which contains the epitope shared with the neurofibrillary tangle, is partially insoluble under aqueous conditions, probably due to the aggregation of oppositely charged residues. Thus, rapid cleavage of MAP2 to small peptides is probably necessary in vivo to prevent the aggregation of larger cleavage fragments.
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PMID:Partial sequence of MAP2 in the region of a shared epitope with Alzheimer neurofibrillary tangles. 245 76

Three-dimensional reconstruction and ultrastructural studies of classical plaques from the cortex of patients with Alzheimer's disease showed that microglial cells of the plaques are the amyloid-forming cells. The amyloid star of the single plaque represents the product of five or six microglial cells covering about 80% of the amyloid star surface. The amyloid fibers appear to be formed within altered cisterns of the endoplasmic reticulum. Distended cisterns form channels filled with amyloid fibers. Numerous vesicles derived from the Golgi apparatus appear to be attached to or fused with the amyloid-filled channels. Reconstruction of the amyloid star and the microglia cell pole that forms the amyloid star reveals three different zones of distribution of cytoplasmic organelles and amyloid deposits. The peripheral zone comprises channels filled with loosely packed amyloid fibers arranged in a parallel manner. The transient zone consists of a mixture of fusing amyloid channels and products of disintegration of cytoplasmic pockets, dense bodies and fragments of cellular membranes. The core of the amyloid star is composed of condensed, densely packed amyloid fibers that are free of cellular debris. Formation of the three zones supports the idea that the microglia/macrophages are not phagocytes but instead are the cells manufacturing the amyloid fibers.
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PMID:Ultrastructural studies of the cells forming amyloid fibers in classical plaques. 255 31

The blood-brain barrier separates brain interstitial space from blood and is formed by brain capillary endothelial cells that are fused together by epithelial-like tight junctions. Study of the blood-brain barrier traditionally has been a relatively arcane field, even for neurobiologists. However, advances over the last 10 years in understanding the transport physiology and cell biology of the brain capillary endothelial cell now provide insights into the pathogenesis of such problems as brain glucopenia, hepatic encephalopathy, therapeutic efficacy of alpha-methyldopa, brain edema in diabetic ketoacidosis, Alzheimer's disease, brain tumors, and lupus cerebritis.
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PMID:Blood-brain barrier: interface between internal medicine and the brain. 287 46

Galanin (GAL) is a biologically active neuropeptide that has been suggested to play a role in stress-induced inhibition of insulin secretion, in dementia of the Alzheimer's type, and in the regulation of growth hormone secretion. We report here the isolation of a bovine genomic clone containing more than 5-kb 5'-flanking sequences. Partial sequence analysis of the genomic clone revealed an atypical TATA-box in the promoter (ATAAATA) and several consensus sequences that typically bind transcription factors, including those that bind NF kappa B, Sp1, and AP-2. Primer extension and RNase protection analyses revealed that transcription is initiated at two sites, 28 and 31 bp, respectively, downstream from the TATA-box. To locate functionally active regulatory elements on the GAL gene, we first identified a neural crest-derived human neuroblastoma cell line, SK-N-SH subclone SH-SY5Y, that expressed easily detectable levels of endogenous GAL mRNA. We then constructed plasmids containing various lengths of bovine GAL 5'-flanking sequences and the first exon fused to a reporter plasmid encoding luciferase. Transfection of these plasmids into the SH-SY5Y cells and analysis by transient expression indicated that 131 bp of 5' gene sequence was sufficient to obtain maximal basal expression. Further, expression was suppressed 16-fold when 5 kb were included, suggesting the presence of a distal repressor element(s). In another set of experiments, we found that GAL mRNA levels could be induced more than 10-fold by 20-hr treatment with phorbol 12-myristate 13-acetate (PMA). In cells transfected with the same plasmids, luciferase activity was also induced by PMA, but the degree of induction did not significantly differ among the deletion constructions (varying from six- to eight-fold), suggesting that elements conferring PMA induction and/or RNA stabilization may be located within 131 bp of the transcriptional start site, in the first exon, or on gene sequences not studied here.
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PMID:Primary sequence and functional analysis of the bovine galanin gene promoter in human neuroblastoma cells. 752 Jul 3

Overexpression of the beta-amyloid precursor protein gene (beta-APP) may contribute to the abnormal generation of beta-amyloid protein in Alzheimer's disease. We demonstrate using a human glial cell line (1321N1) that activation of protein kinase C (PKC) with phorbol 12-myristate 13-acetate (PMA) increases beta-APP mRNA levels, induces known components of the transcription factor activator protein-1 (AP-1), and increases protein-DNA binding activity to AP-1 sequences within the beta-APP promoter. A beta-APP promoter-luciferase reporter gene is transcriptionally activated by PMA, as well as by expression of constitutively activated PKC or by expression of c-Jun. Further characterization suggests that the distal but not the proximal AP-1 recognition site binds nuclear proteins regulated by PKC, and that the AP-1 binding activity is likely to be composed of Jun-Jun homodimers rather than Jun-Fos heterodimers. Additional studies demonstrate that a single copy of the distal AP-1 site fused to a heterologous promoter is sufficient to confer a response to PMA. Mutagenesis of this site in the beta-APP promoter renders it unresponsive to c-Jun and attenuates transcriptional activation by PMA. We suggest that cellular mediators that activate PKC, particularly those that induce significant increases in c-Jun, may up-regulate expression of the beta-APP gene and consequently affect production and processing of this protein.
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PMID:A direct role for protein kinase C and the transcription factor Jun/AP-1 in the regulation of the Alzheimer's beta-amyloid precursor protein gene. 806 12

Amyloid beta protein (A beta P) is the 40-42 residue polypeptide implicated in the pathogenesis of Alzheimer's disease (AD). We have reconstituted this peptide into phosphatidylserine liposomes and then fused the liposomes with a planar lipid bilayer. When incorporated into this bilayer, the A beta P forms cation selective channels capable of transporting calcium and some monovalent cations including cesium, lithium, potassium, and sodium. The channels behave in an ohmic fashion and single channels can be shown to exhibit multiple subconductance states. Hitherto, A beta P has been presumed to be neurotoxic, although direct demonstration of toxicity has proved elusive. On the basis of the present data we suggest that the ion channel activity of the polypeptide may be the basis of its neurotoxic effects.
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PMID:A new hypothesis for the mechanism of amyloid toxicity, based on the calcium channel activity of amyloid beta protein (A beta P) in phospholipid bilayer membranes. 823 77

Amyloid beta protein (A beta P) is the 40- to 42-residue polypeptide implicated in the pathogenesis of Alzheimer disease. We have incorporated this peptide into phosphatidylserine liposomes and then fused the liposomes with a planar bilayer. When incorporated into bilayers the A beta P forms channels, which generate linear current-voltage relationships in symmetrical solutions. A permeability ratio, PK/PCl, of 11 for the open A beta P channel was estimated from the reversal potential of the channel current in asymmetrical KCl solutions. The permeability sequence for different cations, estimated from the reversal potential of the A beta P-channel current for each system of asymmetrical solutions, is Pcs > PLi > PCa > or = PK > PNa. A beta P-channel current (either CS+ or Ca2+ as charge carriers) is blocked reversibly by tromethamine (millimolar range) and irreversibly by Al3+ (micromolar range). The inhibition of the A beta P-channel current by these two substances depends on transmembrane potential, suggesting that the mechanism of blockade involves direct interaction between tromethamine (or Al3+) and sites within the A beta P channel. Hitherto, A beta P has been presumed to be neurotoxic. On the basis of the present data we suggest that the channel activity of the polypeptide may be responsible for some or all of its neurotoxic effects. We further propose that a useful strategy for drug discovery for treatment of Alzheimer disease may include screening compounds for their ability to block or otherwise modify A beta P channels.
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PMID:Alzheimer disease amyloid beta protein forms calcium channels in bilayer membranes: blockade by tromethamine and aluminum. 838 Jun 42

The kinetics of amyloid fibril formation by beta-amyloid peptide (Abeta) are typical of a nucleation-dependent polymerization mechanism. This type of mechanism suggests that the study of the interaction of Abeta with itself can provide some valuable insights into Alzheimer disease amyloidosis. Interaction of Abeta with itself was explored with the yeast two-hybrid system. Fusion proteins were created by linking the Abeta fragment to a LexA DNA-binding domain (bait) and also to a B42 transactivation domain (prey). Protein-protein interactions were measured by expression of these fusion proteins in Saccharomyces cerevisiae harboring lacZ (beta-galactosidase) and LEU2 (leucine utilization) genes under the control of LexA-dependent operators. This approach suggests that the Abeta molecule is capable of interacting with itself in vivo in the yeast cell nucleus. LexA protein fused to the Drosophila protein bicoid (LexA-bicoid) failed to interact with the B42 fragment fused to Abeta, indicating that the observed Abeta-Abeta interaction was specific. Specificity was further shown by the finding that no significant interaction was observed in yeast expressing LexA-Abeta bait when the B42 transactivation domain was fused to an Abeta fragment with Phe-Phe at residues 19 and 20 replaced by Thr-Thr (AbetaTT), a finding that is consistent with in vitro observations made by others. Moreover, when a peptide fragment bearing this substitution was mixed with native Abeta-(1-40), it inhibited formation of fibrils in vitro as examined by electron microscopy. The findings presented in this paper suggest that the two-hybrid system can be used to study the interaction of Abeta monomers and to define the peptide sequences that may be important in nucleation-dependent aggregation.
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PMID:Two-hybrid system as a model to study the interaction of beta-amyloid peptide monomers. 870 Aug 86


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