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  • SEQUENCE  (4)
  • 1
    Keywords: SPECTRA ; CELLS ; EXPRESSION ; CELL ; COMBINATION ; Germany ; human ; PERFUSION ; PROTEIN ; PROTEINS ; SEQUENCE ; SIGNAL ; cell culture ; culture ; ACID ; FORM ; ASSAY ; PURIFICATION ; GOLGI-APPARATUS ; CHROMATOGRAPHY ; HIGH-LEVEL EXPRESSION ; CORE PROTEIN ; serine ; QUANTITIES ; AFFINITY ; PROTEOGLYCAN ; AFFINITY-CHROMATOGRAPHY ; BETA-D-XYLOSYLTRANSFERASE ; glycosyltransferase,proteoglycan,perfusion chromatography,insect cells ; HIGH-LEVEL ; JAR CHORIOCARCINOMA CELLS ; MOLECULAR-CLONING ; PERFUSION CHROMATOGRAPHY ; PROTEIN LINKAGE REGION ; SERUM XYLOSYLTRANSFERASE ; SYSTEMIC-SCLEROSIS ; UDP-D-XYLOSE
    Abstract: Human xylosyltransferase I (XT-I) catalyzes the transfer of xylose from UDP-xylose to consensus serine residues of proteoglycan core proteins. Expression of a soluble form of recombinant histidine-tagged XT-I (rXT-I-HIS) was accomplished at a high level with High Five/pCG255-1 insect cells in suspension culture. The recombinant protein was purified to homogeneity by a combination of heparin affinity chromatography and metal (Ni2+) chelate affinity chromatography. Using the modern technique of perfusion chromatography, a rapid procedure for purification of the rXT-I-HIS from insect cell culture supernatant was developed. The purified, biologically active enzyme was homogeneous on SIDS-PAGE, was detected with anti-XT-I-antibodies, and had the expected tryptic fragment mass spectrum. N-terminal amino acid sequencing demonstrated that the N-terminal signal sequence of the expressed protein was quantitatively cleaved. The total yield of the enzyme after purification was 18% and resulted in a specific XT-I activity of 7.9 mU/mg. The K-m of the enzyme for recombinant [Val(36) Val(38)](delta1),[Gly(92),Ile(94)](delta2) bikunin was 0.8 muM. About 5 rag purified enzyme could be obtained from 1 L cell culture supernatant. The availability of substantial quantities of active, homogeneous enzyme will be of help in future biochemical and biophysical characterization of XT-I and for the development of a immunological XT-I assay. (C) 2003 Elsevier Inc. All rights reserved
    Type of Publication: Journal article published
    PubMed ID: 14680799
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  • 2
    Keywords: CELLS ; EXPRESSION ; Germany ; human ; CDNA CLONES ; GENE ; GENES ; GENOME ; HYBRIDIZATION ; PROTEIN ; PROTEINS ; transcription ; data mining ; radiation ; murine ; FAMILY ; MEMBER ; SEQUENCE ; TRANSPORT ; MOUSE ; IN-SITU ; YEAST ; MEMBRANE ; REQUIRES ; AAA family ; DATABASE ; DISPLAY ; endocytosis ; ENDOSOMAL TRAFFICKING ; FISH ; FUSION PROTEINS ; HUMAN GENOME ; INTRACELLULAR PROTEIN TRAFFICKING ; LATE-GOLGI ; MOUSE SKD1 ; MULTIVESICULAR BODY ; TRAFFICKING ; vacuolar protein sorting ; VPS4- paralogue
    Abstract: The VPS4 gene is a member of the AAA-family; it codes for an ATPase which is involved in lysosomal/endosomal membrane trafficking. VPS4 genes are present in virtually all eukaryotes. Exhaustive data mining of all available genomic databases from completely or partially sequenced organisms revealed the existence of up to three paralogues, VPS4a, -b, and -c. Whereas in the genome of lower eukaryotes like yeast only one VPS4 representative is present, we found that mammals harbour two paralogues, VPS4a and VPS4b. Most interestingly, the Fugu fish contains a third VPS4 paralogue (VPS4c). Sequence comparison of the three VPS4 paralogues indicates that the Fugu VPS4c displays sequence features intermediate between VPS4a and VPS4b. Using complete mammalian VPS4a and VPS4b cDNA clones as probes, genomic clones of both VPS4 paralogues in human and mouse were identified and sequenced. The chromosomal loci of all four VPS4 genes were determined by independent methods. A BLAST search of the human genome database with the human VPS4A sequence yielded a double match, most likely due to a faulty assembly of sequence contigs in the human draft sequence. Fluorescent in situ hybridization and radiation hybrid analyses demonstrated that human and mouse VPS4A/a and VPS4B/b are located on syntenic chromosomal regions. Northern blot and semi-quantitative reverse transcription analyses showed that mouse VPS4a and VPS4b are differentially expressed in different organs, suggesting that the two paralogues have developed different functional properties since their divergence. To investigate the subcellular distribution of the murine VPS4 paralogues, we transiently expressed various fluorescent VPS4 fusion proteins in mouse 3T3 cells. All tested VPS4 fusion proteins were found in the cytosol. Expression of dominant- negative mutant VPS4 fusion proteins led to their concentration in the perinuclear region. Co-expression of VPS4a-GFP and VPS4b-dsRed fusion proteins revealed a partial co-localization that was most prominent with mutant VPS4a and VPS4b proteins. A physical interaction between the mouse paralogues was also supported by two-hybrid analyses. (C) 2003 Elsevier Science B.V. All rights reserved
    Type of Publication: Journal article published
    PubMed ID: 12594041
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  • 3
    Keywords: Germany ; imaging ; SYSTEM ; SYSTEMS ; RESOLUTION ; TIME ; MARKER ; MR ; SEQUENCE ; SEQUENCES ; SIGNAL ; SUSCEPTIBILITY ; FIELD ; MAGNETIC-RESONANCE ; ACQUISITION ; RELAXATION ; TRACKING ; BIOPSY ; RE ; INTERVENTIONAL DEVICES ; INFUSION ; ENHANCEMENT ; SIZE ; COILS ; technique ; VIEW ; RARE ; CATHETERS ; DEFT ; device localization ; fast MRI ; INNER VOLUME ; inner volume imaging ; local look (LoLo) ; percutaneous intervention ; WIRES
    Abstract: Percutaneous MR-guided interventions with needles require fast pulse sequences to image the needle trajectory with minimal susceptibility artifacts. Spin-echo pulse sequences are well suited for reducing artifact size; however, even with single-shot turbo spin-echo techniques, such as rapid acquisition with relaxation enhancement (RARE) or half-Fourier acquisition single-shot turbo spin-echo (HASTE), fast imaging remains challenging. In this work we present a HASTE pulse sequence that is combined with inner-volume excitation to reduce the scan time and limit the imaging field of view (FOV) to a small strip close to the needle trajectory (targeted-HASTE). To compensate for signal saturation from fast repeated acquisitions, a magnetization restore pulse (driven equilibrium Fourier transform (DEFT)) is used. The sequence is combined with dedicated active marker coils to measure the position and orientation of the needle so that the targeted-HASTE image slice is automatically repositioned. In an animal experiment the coils were attached to an MR-compatible robotic assistance system for MR-guided interventions. Needle insertion and infusion via the needle could be visualized with a temporal resolution of 1 s, and the needle tip could be localized even in the presence of a stainless steel mandrel
    Type of Publication: Journal article published
    PubMed ID: 16795081
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  • 4
    Keywords: PEPTIDE ; ENDOTHELIAL-CELLS ; GROWTH ; GROWTH-FACTOR ; IN-VITRO ; INHIBITOR ; Germany ; VITRO ; PROTEIN ; RELEASE ; SEQUENCE ; ASSAY ; TIME-OF-FLIGHT ; EXTRACELLULAR-MATRIX ; BETA-D-XYLOSYLTRANSFERASE ; JAR CHORIOCARCINOMA CELLS ; UDP-D-XYLOSE ; MASSES ; END ; HEPARAN-SULFATE PROTEOGLYCANS ; heparin ; basic fibroblast growth factor ; FACTOR BINDS ; MITOGENIC ACTIVITY ; NONENZYMATIC GLYCOSYLATION ; xylosyltransferase
    Abstract: Human basic fibroblast growth factor (bFGF) is a heparin-binding growth factor containing a G-S-G-motif which is a potential recognition sequence of xylosyltransferase I (XT-I). Here, we show that the recombinant human bFGF was xylosylated in vitro by human XT-I and that the fragment bFGF (1-24) is a good XT-I acceptor (K-m = 20.8 mu M for native XT-I and K-m = 22.3 mu M for recombinant XT-I). MALDI and MALDI-PSD time-of-flight mass spectrometric analyses of the xylosylated bFGF protein demonstrate the transfer of xylose to the serine residue of the G-S-G-motif in the amino terminal end of bFGF. The peptide bFGF (1-24) is well suitable as an acceptor substrate for XT-I and can be used in a radiochemical assay to measure the XT-I activity in cell culture supernatant and human body fluids, respectively. Furthermore; we could demonstrate that the XT-I interacts strongly with heparin and that this glycosaminoglycan is a predominantly non-competitive inhibitor of the enzyme using the fragment bFGF (1-24) as xylose acceptor. (c) 2005 Elsevier Inc. All rights reserved
    Type of Publication: Journal article published
    PubMed ID: 15936726
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