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Dido Vassilacopoulou James A. Ripellino Nikolaos Tezapsidis Vivian Y. H. Hook Nikolaos K. Robakis 《Journal of neurochemistry》1995,64(5):2140-2146
Abstract: The amyloid β peptide (Aβ) of Alzheimer disease is derived from the proteolytic processing of the amyloid precursor proteins (APPs), which are considered type I transmembrane proteins. Here we report that the soluble fraction of isolated adrenal medullary chromaffin granules (CG), a model neuronal secretory vesicle system, contains an antigen that immunochemically and on sodium dodecyl sulfate-polyacrylamide gel electrophoresis was indistinguishable from full-length APP. A truncated APP fragment with intact Aβ sequence was also detected in the soluble fraction of CG. In vitro experiments showed that full-length APP was solubilized from CG membranes at 37°C as a function of pH, with a peak of activity between pH 8.5 and pH 9.0. Solubilization of full-length APP was inhibited by several protease inhibitors, including aprotinin, cystatin, and iodoacetamide, by the divalent cations Ca2+ and Zn2+ , and by preheating of the membranes. These results are consistent with and suggest the involvement of an enzymatic mechanism in the solubilization of potentially amyloidogenic full-length APP. Production of Aβ from a transmembrane APP predicts a proteolytic cleavage within the lipid bilayer, a site relatively inaccessible to proteases. Thus, the detected soluble, potentially amyloidogenic, full-length APP may be a substrate for the proteases producing Aβ. The detection of soluble APP with intact Aβ sequence in secretory vesicles is consistent with the extracellular topology of amyloid depositions. 相似文献
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Volkhard Seitz Sigrid Schaper Anja Dr?ge Dido Lenze Michael Hummel Steffen Hennig 《Nucleic acids research》2015,43(20):e135
Two-step PCR procedures are an efficient and well established way to generate amplicon libraries for NGS sequencing. However, there is a high risk of cross-contamination by carry-over of amplicons from first to second amplification rounds, potentially leading to severe misinterpretation of results. Here we describe a new method able to prevent and/or to identify carry-over contaminations by introducing the K-box, a series of three synergistically acting short sequence elements. Our K-boxes are composed of (i) K1 sequences for suppression of contaminations, (ii) K2 sequences for detection of possible residual contaminations and (iii) S sequences acting as separators to avoid amplification bias. In order to demonstrate the effectiveness of our method we analyzed two-step PCR NGS libraries derived from a multiplex PCR system for detection of T-cell receptor beta gene rearrangements. We used this system since it is of high clinical relevance and may be affected by very low amounts of contaminations. Spike-in contaminations are effectively blocked by the K-box even at high rates as demonstrated by ultra-deep sequencing of the amplicons. Thus, we recommend implementation of the K-box in two-step PCR-based NGS systems for research and diagnostic applications demanding high sensitivity and accuracy. 相似文献
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Boris V. Skryabin Joachim Kremerskothen Dido Vassilacopoulou Todd R. Disotell Vladimir V. Kapitonov Jerzy Jurka Jürgen Brosius 《Journal of molecular evolution》1998,47(6):677-685
The gene encoding BC200 RNA arose from a monomeric Alu element. Subsequently, the RNA had been recruited or exapted into
a function of the nervous system. Here we confirm the presence of the BC200 gene in several primate species among the Anthropoidea.
The period following the divergence of New World monkeys and Old World monkeys from their common ancestor is characterized
by a significantly higher substitution rate in the examined 5′ flanking region than in the BC200 RNA coding region itself.
Furthermore, the conservation of CpG dimers in the RNA coding region (200 bp) is drastically increased compared to the 5′
flanking region (∼400 bp) over all 12 species examined. Finally, the brain-specific expression pattern of BC200 RNA and its
presence as a ribonucleoprotein particle (RNP) are conserved in Old World and New World monkeys. Our studies indicate that
the gene encoding BC200 RNA was created at least 35–55 million years ago and its presence, mode of expression, and association
with protein(s) as an RNP are under selective pressure.
Received: 1 December 1997 / Accepted: 3 June 1998 相似文献
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An endogenous inhibitor of l-Dopa decarboxylase was identified and purified from human serum. In Triton X-114 partitioning experiments, the inhibitor was recovered in the detergent enriched phase, suggesting a hydrophobic nature. Purification was achieved by means of proteinase K digestion, ammonium sulphate precipitation, phenyl sepharose hydrophobic chromatography and subsequent extraction from a nondenaturing polyacrylamide gel. This purification scheme resulted in the isolation of a single 25 kDa band, bearing l-Dopa decarboxylase inhibitory activity. The purified molecule was found to be resistant to heat and digestion by various proteases. Proteolytic digestion of the purified inhibitor by pronase and aminopeptidase M was achieved only following carboxymethylation. The biological importance of the presence of an l-Dopa decarboxylase activity inhibitor in normal biological fluids remains to be elucidated. The better understanding of the regulation of Ddc enzymatic activity could prove valuable in the clarification of the enzyme’s role in a series of pathological conditions, as well as, in physiological regulatory mechanisms.Dido Vassilacopoulou and Emmanuel G. Fragoulis-Shared senior authorship 相似文献
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Stefi Aikaterina L. Vassilacopoulou Dido Routsi Efthymia Stathopoulos Panagiotis Argyropoulou Aikaterini Skaltsounis Alexios-Leandros Christodoulakis Nikolaos S. 《Journal of Plant Growth Regulation》2021,40(3):1044-1059
Journal of Plant Growth Regulation - Biosynthesis of certain secondary metabolites, as a relief response to the severe environmental stress of the mediterranean summer, is a major reaction of the... 相似文献
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Ioanna?Chalatsa Eleftheria?Nikolouzou Emmanuel?G.?Fragoulis Dido?VassilacopoulouEmail author 《Molecular biology reports》2011,38(2):1005-1011
l-Dopa decarboxylase (DDC) catalyses the decarboxylation of l-Dopa. It has been shown that the DDC gene undergoes alternative splicing within its 5′-untranslated region (UTR), in a tissue-specific
manner, generating identical protein products. The employment of two alternative 5′UTRs is thought to be responsible for tissue-specific
expression of the human DDC mRNA. In this study, we focused on the investigation of the nature of the mRNA expression in human
cell lines of neural and non-neural origin. Our results show the expression of a neural-type DDC mRNA splice variant, lacking
exon 3 in all cell lines studied. Co-expression of the full length non-neural DDC mRNA and the neural-type DDC splice variant
lacking exon 3 was detected in all cell lines. The alternative DDC protein isoform, Alt-DDC, was detected in SH-SY5Y and HeLa
cells. Our findings suggest that the human DDC gene undergoes complex processing, leading to the formation of multiple mRNA
isoforms. The study of the significance of this phenomenon of multiple DDC mRNA isoforms could provide us with new information
leading to the elucidation of the complex biological pathways that the human enzyme is involved in. 相似文献
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L-Dopa Decarboxylase is a pyridoxal 5-phosphate (PLP)-dependent enzyme that catalyses the decarboxylation of L-Dopa to dopamine.
In this study, we investigated the cellular topology of the active human enzyme. Fractionation of membranes from human cell
lines, of neural and non-neural origin, by temperature-induced phase separation in Triton X-114 resulted in the detection
of DDC molecules in all separation phases. Solubilization of membrane-associated DDC was observed in a pH and time-dependent
manner and was affected by divalent cations and protease inhibitors, suggesting the involvement of a possible release mechanism.
The study of the biological properties and function of the solubilization phenomenon described here, as well as, the study
of the membrane-associated enzyme could provide us with new information about the participation of the human L-Dopa decarboxylase
in physiological and aberrant processes. 相似文献
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