UMLS:C0272170 - FACTA Search

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Query: UMLS:C0272170 (SDS)
50,377 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Apolipoprotein E (apoE), a protein genetically linked to the incidence of Alzheimer's disease, forms SDS-stable complexes in vitro with beta-amyloid peptide (Abeta), the primary component of senile plaques. In the present study, we investigated whether apoE was able to bind full-length Abeta precursor protein (APP). Using a maltose-binding-protein-APP fusion protein and human very-low-density lipoprotein (VLDL), we detected an interaction of apoE with APP that was inhibited by Abeta or anti-apoE antibody. Saturation-binding experiments indicated a single binding equilibrium with an apparent 1:1 stoichiometry and a dissociation constant of 15 nM. An interaction was also observed using apoE from cerebrospinal fluid or delipidated VLDL, as well as recombinant apoE. APP.apoE complexes were SDS-stable, and their formation was not inhibited by reducing conditions; however, they were dissociated by SDS under reducing conditions. ApoE.APP complexes formed high-molecular-mass aggregates, and competition experiments suggested that amino acids 14-23 of Abeta are responsible for complex-formation. Finally, no differences were found when studying the interaction of APP with apoE3 or apoE4. Taken together, our results demonstrate that apoE may form stable complexes with the Abeta moiety of APP with characteristics similar to those of complexes formed with isolated Abeta, and suggest the intriguing possibility that apoE-APP interactions may be pathologically relevant in vivo.
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PMID:Apolipoprotein E forms stable complexes with recombinant Alzheimer's disease beta-amyloid precursor protein. 922 43

We have previously identified distinct nuclear and mitochondrial isoforms of dUTPase in human cells, reporting the cDNA sequence of the nuclear isoform (DUT-N). We now report a cDNA corresponding to the mitochondrial isoform (DUT-M). The DUT-M cDNA contains an 252-amino acid open reading frame, encoding a protein with a predicted Mr of 26,704. The amino-terminal region of the protein contains an arginine-rich, 69-residue mitochondrial targeting presequence that is absent in the mature protein. In vitro transcription and translation of the DUT-M cDNA results in the production of a precursor protein with an apparent molecular mass of 31 kDa as judged by SDS-polyacrylamide gel electrophoresis. The DUT-M precursor is enzymatically active and immunoreacts with a dUTPase-specific monoclonal antibody. Mitochondrial import and processing studies demonstrate that the DUT-M precursor is processed into a 23-kDa protein and imported into mitochondria in vitro. Isoelectric focusing experiments demonstrate that the DUT-N has a pI of 6.0, while the processed form of DUT-M has a more basic pI of 8.1, measurements that are in agreement with predicted values. Studies aimed at understanding the expression of these isoforms were performed utilizing quiescent and replicating 34Lu human lung fibroblasts as a model cell culture system. Northern blot analysis, employing an isoform-specific probe, demonstrates that DUT-N and DUT-M are encoded by two distinct mRNA species of 1.1 and 1.4 kilobases, respectively. Western and Northern blot analysis reveal that DUT-M protein and mRNA are expressed in a constitutive fashion, independent of cell cycle phase or proliferation status. In contrast, DUT-N protein and mRNA levels are tightly regulated to coincide with nuclear DNA replication status. Because DUT-N and DUT-M have identical amino acid and cDNA sequences in their overlapping regions, we set out to determine if they were encoded by the same gene. The 5' region of the gene encoding dUTPase was isolated and characterized by a combination of Southern hybridization and DNA sequencing. These analyses demonstrate that the dUTPase isoforms are encoded by the same gene with isoform-specific transcripts arising through the use of alternative 5' exons. This finding represents the first example in humans of alternative 5' exon usage to generate differentially expressed nuclear and mitochondrial specific protein isoforms.
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PMID:The human dUTPase gene encodes both nuclear and mitochondrial isoforms. Differential expression of the isoforms and characterization of a cDNA encoding the mitochondrial species. 922 92

Chromogranin A (CGA) is the most abundant protein of the bovine adrenal medulla and plays an important role as precursor protein of several peptides that act as modulators for endocrine cell secretory activity. Furthermore, it is presumed to play a role in the targeting of peptide hormones and neurotransmitters to granules of the regulated pathway. However, its complete primary structure and proteolytic processing have not yet been identified. This study describes a rapid and efficient procedure for the high yield isolation of bovine CGA and its N-terminal processing products, vasostatin I and II. Using the lysate from bovine adrenal medulla chromaffin granules, the soluble proteins were purified by three consecutive HPLC steps, thereby avoiding the use of buffer solutions. The protein fractions were isolated and characterized by SDS-PAGE and Western blot analysis as well as by mass spectrometry. In the latter analysis, the efficiency of matrix-assisted laser desorption ionization-mass spectrometry (MALDI-MS) was demonstrated, enabling the unequivocal and sensitive characterization of proteins from crude mixtures. Sufficient amounts of pure protein were obtained by the present procedure to form the basis for detailed structural studies by spectroscopic methods and X-ray crystallography.
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PMID:Isolation and identification of intact chromogranin A and two N-terminal processing products, vasostatin I and II, from bovine adrenal medulla chromaffin granules by chromatographic and mass spectrometric methods. 924 25

Some clues suggest that neuronal damage induces a secondary change of amyloid beta protein (Abeta) metabolism. We investigated this possibility by analyzing the secretion of Abeta and processing of its precursor protein (amyloid precursor protein, APP) in an in vitro model of neuronal apoptosis. Primary cultures of rat cerebellar granule neurons were metabolically labeled with [35S]methionine. Apoptosis was induced by shifting extracellular KCl concentration from 25 mM to 5 mM for 6 h. Control and apoptotic neurons were then subjected to depolarization-stimulated secretion. Constitutive and stimulated secretion media and cell lysates were immunoprecipitated with antibodies recognizing regions of Abeta, full-length APP, alpha- and beta-APP secreted forms. Immunoprecipitated proteins were separated by SDS/PAGE and quantitated with a PhosphorImager densitometer. Although intracellular full-length APP was not significantly changed after apoptosis, the monomeric and oligomeric forms of 4-kDa Abeta were 3-fold higher in depolarization-stimulated secretion compared with control neurons. Such increments were paralleled by a corresponding increase of the beta-APPs/alpha-APPs ratio in apoptotic secretion. Immunofluorescence studies performed with an antibody recognizing an epitope located in the Abeta sequence showed that the Abeta signal observed in the cytoplasm and in the Golgi apparatus of control neurons is uniformly redistributed in the condensed cytoplasm of apoptotic cells. These studies indicate that neuronal apoptosis is associated with a significant increase of metabolic products derived from beta-secretase cleavage and suggest that an overproduction of Abeta may be the consequence of neuronal damage from various causes.
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PMID:Increased amyloidogenic secretion in cerebellar granule cells undergoing apoptosis. 944 17

NACP, the precursor protein of the non-amyloid beta/A4 protein (A beta) component of Alzheimer's disease (AD) amyloid, also known as alpha-synuclein, was suggested to seed amyloid plaque formation in AD by stimulating A beta aggregation. We have demonstrated that NACP experienced self-oligomerization only in the presence of a modified A beta fragment (A beta25-35) by using dicyclohexylcarbodiimide. This NACP oligomerization, appearing as a discrete ladder on a Tricine SDS-PAGE, was not observed with other A beta peptides such as the reverse peptide A beta35-25 and A beta1-40, indicating this process was specific not only for the C-terminal peptide sequence of the A beta but also for its orientation. It might be, therefore, suggested that the NACP self-oligomers formed only in the presence of a N-terminally truncated A beta peptide could act as a nucleation center for plaque formation during AD development.
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PMID:Self-oligomerization of NACP, the precursor protein of the non-amyloid beta/A4 protein (A beta) component of Alzheimer's disease amyloid, observed in the presence of a C-terminal A beta fragment (residues 25-35). 946 43

Two PCR-amplified genomic DNA fragments encoding apple (cv. Fuji) polyphenol oxidase (PPO) were cloned and sequenced. A comparison of genomic DNA with cDNAs revealed that the PPOs lacked introns. Both PPO DNAs appear to encode a 66-kDa precursor protein consisting of a 56-kDa mature protein and a N-terminal transit peptide of 10-kDa N-terminal transit peptide. Apple PPO DNA was expressed in Escherichia coli, and the gene product (56 kDa) without a transit peptide was immunochemically detected and was the same size (ca. 65 kDa) as the main PPO of apple fruit by SDS-PAGE.
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PMID:Cloning genomic DNA encoding apple polyphenol oxidase and comparison of the gene product in Escherichia coli and in apple. 953 95

Porphyromonas gingivalis has been implicated as a major aetiological agent in certain forms of periodontal disease, P. gingivalis is a Gram-negative, asaccharolytic bacterium that obtains energy from the fermentation of amino acids derived from the hydrolysis of host protein. Virulence factors of this bacterium include the capsule, fimbrial adhesins, cytotoxins and extracellular hydrolytic enzymes. A 43 kDa fimbrillin from P. gingivalis has been isolated and characterized. However, there is evidence that a second type of fimbria exists on the surface of P. gingivalis. A putative P. gingivalis fimbrial protein from a membrane preparation has been isolated and identified. This protein was shown to be reactive with sera from patients harbouring P. gingivalis. A 28 kDa protein fragment was purified by anion exchange, gel filtration and reversed-phase chromatography. N-terminal sequence analysis of the 28 kDa protein fragment revealed homology to the fimbrial precursor protein of Dichelobacter nodosus. A peptide corresponding to the N-terminal 26 amino acyl residues of the 28 kDa protein fragment was synthesized and used to raise antibodies to the protein. Western blot analysis after SDS-PAGE of a P. gingivalis membrane preparation using the antibodies raised to the synthetic peptide detected three proteins of 36, 41 and 67 kDa. When protease inhibitors were not included in the extraction procedure only the 36 and 41 kDa bands were detected. It would appear, therefore, that the intact protein has an M(r) of 67 kDa and that the 28, 36 and 41 kDa bands represent protein fragments produced by endogenous proteolytic activity. Based on sequence homology, the 67 kDa protein is possibly a sub-unit of a second P. gingivalis fimbrial type or a surface receptor.
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PMID:Purification and characterization of a putative fimbrial protein/receptor of Porphyromonas gingivalis. 961 83

A protein (mCLCA1) has been cloned from a mouse lung cDNA library that bears strong sequence homology with the recently described bovine tracheal, Ca2+-sensitive chloride channel protein (bCLCA1), bovine lung endothelial cell adhesion molecule-1 (Lu-ECAM-1), and the human intestinal Ca2+-sensitive chloride channel protein (hCLCA1). In vitro, its 3.1-kilobase message translates into a 100-kDa protein that can be glycosylated to an approximately 125-kDa product. SDS-polyacrylamide gel electrophoresis from lysates of mCLCA1 cDNA-transfected transformed human embryonic kidney cells (HEK293) reveals proteins of 130, 125, and 90 kDa as well as a protein triplet in the 32-38 kDa size range. Western analyses with antisera raised against Lu-ECAM-1 peptides show that the N-terminal region of the predicted open reading frame is present only in the larger size proteins (i.e. 130, 125, and 90 kDa), whereas the C-terminal region of the open reading frame is observed in the 32-38 kDa size proteins, suggesting a posttranslational, proteolytic processing of a precursor protein (125/130 kDa) into 90 kDa and 32-38 kDa components similar to that reported for Lu-ECAM-1. Hydrophobicity analyses predict four transmembrane domains for the 90-kDa protein. The mCLCA1 mRNA is readily detected by Northern analysis and by in situ hybridization in the respiratory epithelia of trachea and bronchi. Transient expression of mCLCA1 in HEK293 cells was associated with an increase in whole cell Cl- current that could be activated by Ca2+ and ionomycin and inhibited by 4, 4'-diisothiocyanatostilbene-2,2'-disulfonic acid, dithiothreitol, and niflumic acid. The discovery of mCLCA1 opens the door for further investigating the possible contribution of a Ca2+-sensitive chloride conductance to the pathogenesis of cystic fibrosis.
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PMID:Molecular and functional characterization of a calcium-sensitive chloride channel from mouse lung. 982 85

Synthesis, processing and agonist-induced modifications of the V2 vasopressin receptor were examined in stably or transiently transfected HEK293 cells. Metabolic labeling with S methionine for 30 min revealed a predominant precursor protein which subsequently gave rise to the mature receptor on the cell surface. Maturation of the receptor was unrelated to glycosylation suggesting that it was the consequence of protein refolding. In addition to monomeric forms of V2 receptor protein, oligomers of the precursor protein were also detected in SDS-PAGE. These oligomers seemed to be dimers and tetrameres, and were more apparent in transiently transfected cells that produced higher quantities of protein then stably transfected cells. No oligomers of the mature receptor were detected, and co-transfection of the wild type with a mutant V2 receptor lacking G-protein coupling activity did not alter the function of the wild type receptor. These results indicated that the formation of oligomeric was most likely a consequence of overproduction of the protein and not a required step for receptor function. Addition of vasopressin promoted phosphorylation and sequestration of the wild type receptor, and of the R137H mutant receptor which lacks coupling to G proteins. Activation of protein kinases A or C did not result in phosphorylation of un-occupied receptor. Phosphate incorporated into the protein was stable in the continuous presence of the ligand despite sequestration of the receptor protein. Deletion of the last 14 amino acids abolished receptor phosphorylation but not sequestration and desensitization, indicating that these two processes are not dependent on protein phosphorylation. Additionally, phosphorylation and sequestration of the R137H mutant receptor revealed that phosphorylation and sequestration does not require coupling to Gs. The wild type V2 vasopressin receptor was found to be palmitoylated at two cysteines at the carboxyl terminus. Either cysteine could be palmitoylated independently of each other and the presence of at least one was required to obtain receptor expression similar to the wild type. The turnover of the palmitic acid incorporated into the receptor was not altered by the addition of vasopressin demonstrating that this post-translational modification of the receptor was not altered by the ligand-promoted phosphorylation of the protein.
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PMID:Processing and ligand-induced modifications of the V2 vasopressin receptor. 1002 23

The nuclear lamina protein, lamin A is produced by proteolytic cleavage of a 74 kDa precursor protein, prelamin A. The conversion of this precursor to mature lamin A is mediated by a specific endoprotease, prelamin A endoprotease. Subnuclear fractionation indicates that the prelamin A endoprotease is localized at the nuclear membrane. The enzyme appears to be an integral membrane protein, as it can only be removed from the nuclear envelope with detergent. It is effectively solubilized by the detergent n-octyl-beta-D-glucopyranoside and can be partially-purified (approximately 1200-fold) by size exclusion and cation exchange (Mono S) chromatography. Prelamin A endoprotease from HeLa cells was eluted from Mono S with 0.3 M sodium chloride as a single peak of activity. SDS-PAGE analysis of this prelamin A endoprotease preparation shows that it contains one major polypeptide at 65 kDa and smaller amounts of a second 68 kDa polypeptide. Inhibition of the enzyme activity in this preparation by specific serine protease inhibitors is consistent with the enzyme being a serine protease.
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PMID:Subcellular localization and partial purification of prelamin A endoprotease: an enzyme which catalyzes the conversion of farnesylated prelamin A to mature lamin A. 1035 58

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