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  • 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 ; ENDOTHELIAL-CELLS ; EXPRESSION ; INHIBITOR ; Germany ; INHIBITION ; KINASE ; PATHWAYS ; TYROSINE KINASE ; SITE ; SITES ; ACTIVATION ; MECHANISM ; LYMPH-NODES ; mechanisms ; FLOW ; ACTIVATED PROTEIN-KINASE ; PHOSPHORYLATION ; TYROSINE KINASE INHIBITOR ; STIMULATION ; antibodies ; antibody ; CLEAVAGE ; MAP KINASE ; GLUTATHIONE ; Western-blot ; MEMBRANE ; SIGNAL-TRANSDUCTION ; SIGNALING PATHWAY ; SURFACE ; PLASMA-MEMBRANE ; western blot ; CELL-SURFACE ; DEPENDENT REGULATION ; shedding ; CROSS-LINKING ; SURFACE EXPRESSION ; DOWN- REGULATION ; HUMAN NEUTROPHILS ; NEUTRAL SPHINGOMYELINASE
    Abstract: Leukocyte recruitment to lymph nodes or inflammatory sites is regulated by adhesion and activation. L-selectin (CD62L) is expressed on leukocytes and mediates tethering and rolling of leukocytes on endothelial cells. Upon stimulation L-selectin is down-regulated by proteolytic cleavage but the molecular mechanisms regulating this shedding step are poorly defined. To study intracellular mechanisms, we induced shedding of L- selectin by cross-linking with an immobilized L-selectin antibody (Dreg56) in Jurkat cells. The loss of surface expression was quantitated by flow cytometry and the increase of soluble L-selectin was determined by Western blot analysis. We find that Jurkat and p56(Ick)-deficient JCaM1.6 cells released L-selectin to similar extent (18 +/- 4% and 17 +/- 3%, respectively) and revealed comparable inhibition with the src- tyrosine kinase inhibitor PP2. Glutathione (GSH), an inhibitor of the neutral sphingomyelinase, PD98059, a MAP-kinase (MAP-K) inhibitor and metalloprotease inhibitors (MPI) (TAPI, Ro 31- 9790, and BB-3103) reduced significantly L-selectin-induced shedding by 60-80%. In Jurkat cells, L-selectin was present in Triton X-100 insoluble membrane rafts and was constitutively tyr-phosphorylated. Dreg56 cross-linking enhanced phosphorylation and recruitment of L-selectin into rafts which was significantly, decreased by pretreatment of cells with PD98059. We conclude, that the metalloproteinase-mediated cleavage of L-selectin from cell surface is triggered by intracellular signaling pathways that are independent of p56(Ick) tyrosine kinase activity, but require other tyrosine kinases and the neutral sphingomyelinase. The cleavage of L- selectin might involve membrane rafts as signaling platform. (C) 2002 Published by Elsevier Science (USA)
    Type of Publication: Journal article published
    PubMed ID: 12504120
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  • 3
    Keywords: GROWTH ; Germany ; MODEL ; QUANTIFICATION ; screening ; SYSTEM ; PROTEIN ; SAMPLE ; SAMPLES ; EFFICIENCY ; MOLECULAR CHARACTERIZATION ; TISSUE ; ENRICHMENT ; HPLC ; QUANTITATION ; BIOLOGY ; MOLECULAR-BIOLOGY ; PHOSPHORYLATION ; PHOSPHORUS ; STAGE ; mass spectrometry ; MASS-SPECTROMETRY ; PEPTIDES ; LIQUID-CHROMATOGRAPHY ; AFFINITY-CHROMATOGRAPHY ; PROTEOMICS ; PHOSPHOPEPTIDES ; METHIONINE ; neutral loss ; phosphoproteins ; element mass spectrometry ; protein phosphorylation ; signaling ; MASSES ; molecular biology ; molecular ; RE ; ARABIDOPSIS-THALIANA ; EXTENSION ; ICP-MS ; SULFUR ; MS ; ARABIDOPSIS ; development ; LEVEL ; MASS ; EVENTS ; technique ; PHOSPHOPROTEIN ; USA ; function ; CAPILLARIES ; MOTILITY ; ACCESS ; STOICHIOMETRY ; quantitative ; CASCADE ; plant ; algae ; Arabidopsis thaliana ; Chlamydomonas reinhardtii ; MOAC
    Abstract: Many essential cellular functions such as growth rate, motility, and metabolic activity are linked to reversible protein phosphorylation, since they are controlled by signaling cascades based mainly on phosphorylation/dephosphorylation events. Quantification of global or site-specific protein phosphorylation is not straightforward with standard proteomic techniques. The coupling of capillary liquid chromatography (mu LC) with ICP-MS (inductively coupled plasma-mass spectrometry) is a method which allows a quantitative screening of protein extracts for their phosphorus and sulfur content, and thus provides access to the protein phosphorylation degree. In extension of a recent pilot study, we analyzed protein extracts from the model organisms Arabidopsis thaliana and Chlamydomonas reinhardtii as representatives for multicellular and unicellular green photosynthetically active organisms. The results indicate that the average protein phosphorylation level of the algae C reinhardtii is higher than that of A. thaliana. Both the average phosphorylation levels were found to be between the extreme values determined so far for prokaryotes (C glutamicum, lowest levels) and eukaryotes (Mus musculus, highest levels). Tissue samples of A. thaliana representing different stages of plant development showed varying levels of protein phosphorylation indicating a different adjustment of the kinase/phosphatase system. We also utilized the pLC-ICP-MS technology to estimate the efficiency of a novel phosphoprotein enrichment method based on aluminum hydroxide, since the enrichment of phosphorylated species is often an essential step for their molecular characterization. (c) 2007 Elsevier Inc. All rights reserved
    Type of Publication: Journal article published
    PubMed ID: 17288992
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  • 4
    Keywords: ACTIVATION, APOPTOSIS, BIOLOGY, chemosensitivity, DOMAIN, ELEMENT, FAMILIES, FAMILY, FAS/CD95/TNFRSF
    Abstract: p63 and p73 express two main classes of isoforms: isoforms which contain the transactivation domain (TAp73 and TAp63) executing transcriptional activity and dominant-negative isoforms which are truncated at the NH2-terminus acting as operant inhibitors of TAp73, TAp63 and wild-type p53, and thus possessing oncogenic potential. Like wt p53, TAp63 and TAp73 isoforms transactivate target genes that activate apoptosis signaling pathways. In an attempt to understand how the CD95 gene is regulated by the p53 family, we investigated the contributions of a p53-responsive element (RE) within the first intron of the CD95 gene as well as three elements within the promoter. The intronic element conferred transcriptional activation by p53, TAp63 and TAp73 and cooperated with the p53-REs in the promoter of the CD95 gene. Cooperation between the p53-REs in the promoter and the intronic p53-binding site resulted in maximal transcriptional activation of the CD95 gene by the p53 family. (C) 2009 Elsevier Inc. All rights reserved
    Type of Publication: Journal article published
    PubMed ID: 19615968
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  • 5
    Abstract: Systemic primary carnitine deficiency (CDSP, MIM 212140) is a disorder of fatty acid oxidation manifesting in acute metabolic decompensation or in progressive cardiomyopathy and muscle weakness. Mutations in the plasmalemmal organic cation/carnitine transporter OCTN2 were recently identified in CDSP patients of diverse ethnic backgrounds. We have performed OCTN2 mutation analysis in two unrelated German patients with primary carnitine deficiency and identified three molecular abnormalities. On one of the four chromosomes analyzed, we detected an Arg169Gln missense mutation that affects an arginine residue absolutely conserved in the entire transporter superfamily to which OCTN2 belongs. On the three other chromosomes, we found an Arg282ter nonsense mutation in exon 5. This mutation is embedded into different haplotypes of closely spaced intragenic dimorphisms in our two patients and was recently described in a patient of Asiatic Indian background, so it appears to be a recurrent or ancient founder mutation that may account for more CDSP cases. Finally, we found that the Arg282ter nonsense mutation is associated with a splicing abnormality at the intron 6/exon 7 junction. However, no mutations are present in exon 6, intron 6, or exon 7, suggesting that defective splicing of exon 7 on the Arg282ter allele is due to an unconventional, long-distance mechanism.
    Type of Publication: Journal article published
    PubMed ID: 10425211
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  • 6
    Abstract: Multiple cell-cell interactions control bone morphogenesis and vascularization. We have employed a spheroidal coculture system of endothelial cells (EC) and osteoblasts (OB) to study cell contact-dependent gene regulation between these two cell types that may play a role in regulating OB differentiation and EC angiogenic properties. Coculture spheroids differentiate spontaneously to organize into a core of OB and a surface layer of endothelial cells. Individual spheroid culture of EC or OB leads to significant alterations in gene expression compared to standard monolayer culture (upregulation of Tie-2 in EC; upregulation of angiopoietin-2 in osteoblasts). More importantly, spheroidal coculture of endothelial cells and osteoblasts leads to significant changes of gene expression in both cell populations (upregulation of VEGFR-2 in EC; downregulation of VEGF, and upregulation of alkaline phosphatase in osteoblasts). These changes are dependent on cell-cell contact and are not seen in stimulation experiments with conditioned supernatants. Collectively, the data demonstrate complex bi-directional gene regulation mechanisms between EC and OB that are likely to play a critical role during OB differentiation and in controlling the properties of angiogenic EC.
    Type of Publication: Journal article published
    PubMed ID: 15325284
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  • 7
    Keywords: IN-VIVO ; RNA ; COMPLEX ; MATURATION ; RECOGNITION ; REGION ; LUCIFERASE ; SUBSET ; MICRORNA ; DICER ; Drosha ; Microprocessor ; miR-16 ; miRNA processing assay
    Abstract: The RNAse III Drosha is responsible for the first step of microRNA maturation, the cleavage of primary miRNA to produce the precursor miRNA. Processing by Drosha is finely regulated and influences the amount of mature microRNA in a cell. We describe in the present work a method to quantify Drosha processing activity in-vivo, which is applicable to any microRNA. With respect to other methods for measuring Drosha activity, our system is faster and scalable, can be used with any cellular system and does not require cell sorting or use of radioactive isotopes. This system is useful to study regulation of Drosha activity in physiological and pathological conditions.
    Type of Publication: Journal article published
    PubMed ID: 21352811
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  • 8
    Keywords: transcriptome ; Illumina ; RNA-SEQ ; Next generation sequencing ; TruSeq RNA ; Strand-specific sequencing ; dUTP
    Abstract: Preserving the original RNA orientation information in RNA-Sequencing (RNA-Seq) experiment is essential to the analysis and understanding of the complexity of mammalian transcriptomes. We describe herein a simple, robust, and time-effective protocol for generating strand-specific RNA-seq libraries suited for the Illumina sequencing platform. We modified the Illumina TruSeq RNA sample preparation by implementing the strand specificity feature using the dUTP method. This protocol uses low amounts of starting material and allows a fast processing within two days. It can be easily implemented and requires only few additional reagents to the original Illumina kit.
    Type of Publication: Journal article published
    PubMed ID: 22609201
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  • 9
    Keywords: CANCER ; EXPRESSION ; INHIBITION ; MICE ; 3T3-L1 ADIPOCYTES ; AMELIORATION
    Abstract: Resveratrol is identified as polyphenolic compound with anti-inflammatory, antioxidant, anti-insulin resistance characteristics. Moreover, resveratrol exerts pro-apoptotic effects in varieties of cancer cell lines. However, effects and mechanisms of resveratrol on the regulation of adipocytes apoptosis remain largely unknown. In this study, we found that resveratrol treatment could induce cell apoptosis in murine 3T3-L1 adipocytes. Furthermore, resveratrol activated the mitochondrial apoptotic signaling pathway with the decrease in the mitochondrial membrane potential (MMP), and the activation of caspase 3. Mechanistically, we found that phosphorylation level of AMP-activated protein kinase alpha (AMPKalpha) was elevated, accompany with reduced level of phosphorylation of protein kinase B (AKT) when cells were exposed to resveratrol. By using small interfering RNAs of AMPKalpha and specific inhibitor for p-AKT, it was shown that activation of AMPKalpha could inhibit downstream of p-AKT, consequently activating mitochondrion-mediated apoptotic pathway. Additionally, we observed similar pro-apoptotic effects of Res on mouse primary adipocytes. Our findings clarified the apoptotic effects and underlying mechanisms of resveratrol in adipocytes, suggesting its potential therapeutic application in the treatment or prevention of obesity and related metabolic symptoms.
    Type of Publication: Journal article published
    PubMed ID: 25603053
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  • 10
    Abstract: Separase is a caspase-like cysteine protease that is best known for its essential role during the metaphase-to-anaphase transition when it cleaves the cohesin ring complex that keeps the sister chromatids together. Another important function of separase is to regulate the process of centriole separation, known as centriole disengagement, at the end of mitosis. We used proximity-dependent biotin identification (BioID) to expand our knowledge on the identity of separase's proximity interactors. We show that separase BioID labeled two domains at the mother centriole: an area underneath the centriolar appendages and another at the proximal end of the mother centriole. BioID analysis identified more than 200 proximity interactors of separase, one being the Alstrom Syndrome Protein 1 (ALMS1) at the base of centrioles. Other proximity interactors are the histone chaperons NAP1L1 and NAP1L4, which localize to the spindle poles during mitosis and the spindle assembly checkpoint proteins BUBR1, SKA1 and SKA3 that reside at kinetochores in early mitosis. Finally, we show that depletion of BUBR1 homolog from Caenorhabditis elegans delayed the recruitment of separase to mitotic chromosomes, and eventually anaphase onset.
    Type of Publication: Journal article published
    PubMed ID: 27495871
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