Your email was sent successfully. Check your inbox.

An error occurred while sending the email. Please try again.

Proceed reservation?

Export
  • 1
    Keywords: ENERGIES ; Germany ; SYSTEM ; PROTEIN ; RESOLUTION ; TIME ; COMPLEX ; COMPLEXES ; DONOR ; BIOLOGY ; MOLECULAR-BIOLOGY ; SEQUENCE ; SEQUENCES ; SPECTROSCOPY ; ENERGY ; US ; CRYSTAL-STRUCTURE ; STABILITY ; FLUORESCENCE ; CHROMATIN STRUCTURE ; ANGSTROM RESOLUTION ; CORRELATION SPECTROSCOPY ; SUBPOPULATION ; molecular biology ; molecular ; CHEMISTRY ; RE ; LEVEL ; methods ; USA ; single-molecule spectroscopy ; nucleosome positioning ; RESONANCE ; CORE PARTICLE ; ACCESSIBILITY ; DNA TARGET SITES ; fluorophore stability ; HISTONE OCTAMER ; MOLECULE ANALYSIS ; nucleosome reconstitution ; nucleosome remodeling ; single-pair FRET ; TRANSCRIBING POLYMERASE
    Abstract: We applied fluorescence detection methods on the single-molecule level to study structural variations and dynamic processes occurring within nucleosomes. Four fluorescent nucleosome constructs were made by attaching donor and acceptor fluorophores to different positions of two nucleosome positioning sequences and reconstituting nucleosornes by salt dialysis. The photochemical and biochemical stability of nucleosomes under single-molecule conditions was optimized by adding inert protein and free radical capturing additives, allowing us to define the best experimental conditions for single-molecule spectroscopy on highly diluted solutions of nucleosome complexes. We could demonstrate for the first time the resolution of conformational subpopulations of nUcleosornes by single-pair fluorescence resonance energy transfer in a freely diffusing system and could show the effect of thermally induced nucleosome repositioning. (c) 2007 Elsevier Inc. All rights reserved
    Type of Publication: Journal article published
    PubMed ID: 17553453
    Signatur Availability
    BibTip Others were also interested in ...
  • 2
    Keywords: EXPRESSION ; IN-VITRO ; Germany ; KINASE ; VITRO ; SITES ; PROTEIN ; COMPLEX ; BIOLOGY ; PHOSPHORYLATION ; PROTEIN-KINASE ; CLEAVAGE ; PERFORMANCE ; IDENTIFICATION ; mass spectrometry ; MASS-SPECTROMETRY ; AFFINITY ; PHOSPHOPEPTIDES ; neutral loss ; PROTEIN-PHOSPHORYLATION ; molecular biology ; CHEMISTRY ; PHOSPHORYLATION SITES ; SUBSTRATE ; Plasmodium falciparum ; proteases ; USA ; PROTEOLYSIS ; PfCDPK1 ; Plasmodium ; Liquid chromatography-tandem mass spectrometry ; DIGESTION ; IMAC ; Hierarchical phosphorylation ; Interference of phosphorylation with proteolysis ; Mutually exclusive phosphorylation ; PfGAP45
    Abstract: Plasmodium falciparum glideosome-associated protein 45 (PfGAP45) was in vitro phosphorylated by P. falciparum calcium-dependent protein kinase (PfCDPK1) and digested using the four proteases trypsin, chymotrypsin, AspN, and elastase. Subsequently, phosphopeptide enrichment using Ga(III) immobilized metal affinity chromatography (IMAC) was performed. The resulting fractions were analyzed using ultra performance liquid chromatography-electrospray ionization-tandem mass spectrometry (UPLC-ESI-MS/MS), resulting in the identification of a total of nine phosphorylation sites: Ser31, Ser89, Ser103, Ser109, Ser121, Ser149, Ser156, Thr158, and Ser173. During in-depth analyses of the detected phosphopeptides, it was observed that phosphorylation alters the properties of PfGAP45 as kinase and protease substrate. The closely adjacent phosphorylation sites Ser156 (major site) and Thr158 (minor site) were analyzed in detail because at first glance the specific proteases gave highly variable results with respect to the relative abundance of these sites. It was observed that (i) formation of pSer156 and pThr158 was mutually exclusive and (ii) phosphorylation at Ser156 or Thr158 interfered specifically with proteolysis by chymotrypsin or trypsin, respectively. The latter effect was studied in detail using synthetic phosphopeptides carrying either pSer156 or pThr158 as substrate for chymotrypsin or trypsin, respectively. (C) 2009 Elsevier Inc. All rights reserved
    Type of Publication: Journal article published
    PubMed ID: 19549500
    Signatur Availability
    BibTip Others were also interested in ...
  • 3
    Keywords: CLONING ; YEAST ; GENETIC SELECTION
    Abstract: The yeast one-hybrid system (1H-system) is applied to studies of transcription-linked DNA-protein interactions under in vivo conditions detected by reporter gene expression. By use of a variant of green fluorescent protein (GFP) as a reporter, the new 1H-system generation represents a time- and work-saving alternative to the established HIS3/lacZ-based form. However, hitherto, a positive control proving the functionality of the system has been missing. We designed a corresponding control vector combination by subcloning mouse p53 cDNA and its binding sequence and, to get the system working, modified the distance between cloning site and reporter gene promoter in the reporter vector by site-directed mutagenesis. This provides, for the first time, a positive control vector combination for the GFP 1H-system, crucial for its employment in any DNA-protein interaction studies.
    Type of Publication: Journal article published
    PubMed ID: 11969195
    Signatur Availability
    BibTip Others were also interested in ...
  • 4
    Keywords: RECEPTOR ; GROWTH ; ACTIVATION ; BINDING ; SPECTROSCOPY ; PERFORMANCE ; LECTIN ; SUGAR CODE ; GLYCOPROTEINS ; MEMBRANE L1
    Abstract: Phosphorylation is known to have a strong impact on protein functions. We analyzed members of the lectin family of multifunctional galectins as targets of the protein kinases CK1, CK2, and PKA. Galectins are potent growth regulators able to bind both glycan and peptide motifs at intra- and extracellular sites. Performing in vitro kinase assays, galectin phosphorylation was detected by phosphoprotein staining and autoradiography. The insertion of phosphoryl groups varied to a large extent depending on the type of kinase applied and the respective galectin substrate. Sites of phosphorylation observed in the recombinant galectins were determined by a strategic combination of phosphopeptide enrichment and nano-ultra-performance liquid chromatography tandem mass spectrometry (nanoUPLC-MS/MS). By in silico modeling, phosphorylation sites were visualized three-dimensionally. Our results reveal galectin-type-specific Ser-/Thr-dependent phosphorylation beyond the known example of galectin-3. These data are the basis for functional studies and also illustrate the analytical sensitivity of the applied methods for further work on human lectins.
    Type of Publication: Journal article published
    PubMed ID: 24333252
    Signatur Availability
    BibTip Others were also interested in ...
  • 5
    Keywords: PEPTIDE ; SPECTRA ; Germany ; PROTEIN ; BIOLOGY ; MOLECULAR-BIOLOGY ; FUSION ; PEPTIDES ; SECONDARY STRUCTURE ; molecular biology ; molecular ; CHEMISTRY ; methods ; USA ; SPECTRUM
    Type of Publication: Journal article published
    PubMed ID: 17320030
    Signatur Availability
    BibTip Others were also interested in ...
  • 6
    Keywords: human ; KINASE ; SITE ; SITES ; PROTEIN ; PHOSPHORYLATION ; SEQUENCE ; SUBUNIT ; PLASMA ; mass spectrometry ; PEPTIDES ; SPECT ; AFFINITY-CHROMATOGRAPHY ; PHOSPHOPEPTIDES ; PHOSPHOPROTEOME ; SELECTIVE DETECTION ; PROTEIN-PHOSPHORYLATION ; fetuin ; fibrinogen ; INVITRO ; LC-ICP-MS phosphorus-31 ; phosphoproteins
    Abstract: We have used one-dimensional polyacrylamide get electrophoresis, tryptic digestion, and capillary liquid chromatography-mass spectrometry with inductively coupled plasma ionization and phosphorus-31 detection or electrospray ionization for the analysis of protein phosphorylation. We have analyzed human fibrinogen with two well-characterized phosphorylation sites and bovine fetuin with unknown phosphorylation status. Both serine-3 and serine-345 (both in Aalpha) of fibrinogen were clearly recognized. In bovine fetuin. four phosphorylated sites were newly characterized (serine-138, serine-320, serine-323, and serine-324). The novel strategy provides a fast and quantitative overview of the presence of protein phosphorylation sites. (C) 2003 Elsevier Science (USA). All rights reserved
    Type of Publication: Journal article published
    PubMed ID: 12729597
    Signatur Availability
    BibTip Others were also interested in ...
  • 7
    Keywords: CELLS ; EXPRESSION ; CELL ; evaluation ; Germany ; human ; IN-VIVO ; VIVO ; SUPPORT ; CDNA ; GENE ; GENES ; RNA ; TISSUE ; MARKER ; metastases ; ABERRATIONS ; EXTRACELLULAR-MATRIX ; CHAIN-REACTION ; microdissection ; FUNCTIONAL GENOMICS ; physiology ; LOCATION ; CDNAS ; MATRIX ; CHAIN ; PATTERN ; GUIDANCE ; cryosectioning ; EXTRACTION ; osteocyte RNA ; transcript profiling ; undecalcified bone
    Abstract: Osteocytes, the most abundant bone cell type with important roles in tissue maintenance and pathological aberrations such as observed in bone metastases, are enclosed within a highly compact, calcified extracellular matrix. This location complicates analysis in native bone, with the consequence that despite their importance their in vivo molecular physiology is only poorly understood. We have examined the possibility of isolating osteocyte RNA for transcript profiling from native, frozen bone instead of employing the formalin-fixed, paraffin-embedded, decalcified version routinely used in histology, providing chemically modified and highly disintegrated RNAs. Bone tissue was tape-assisted cryosectioned and fixed to glass slides by support of UV-flash-triggered adhesive polymerization followed by quick hematoxylin-eosin staining to generate a guidance image for microdissection. Using an UVa-nitrogen laser, matrix-enclosed osteocytes were either excised and catapulted into RNA preparation vials or freed of accompanying nonosteocyte cellular material. The influences of bone sectioning, staining, and osteocyte capturing procedures on the prepared osteocyte RNAs were analyzed and the method was optimized accordingly. The obtained osteocyte RNAs showed the expected expression pattern of marker genes (reverse transcriptase-polymerase chain reaction), and, following conversion into fluorescent-labeled cDNAs, led to transcript profiles (cDNAchips; 2600 genes) with scatter-graph geometries indicating suitability for high-confidence evaluation. With the approach described here we introduce a methodological way for the characterization of the in vivo molecular physiology of osteocytes by functional genomics. (C) 2004 Elsevier Inc. All rights reserved
    Type of Publication: Journal article published
    PubMed ID: 15556565
    Signatur Availability
    BibTip Others were also interested in ...
  • 8
    Keywords: EXPRESSION ; tumor ; carcinoma ; CELL ; Germany ; COMMON ; GENE ; GENES ; transcription ; TISSUE ; TUMORS ; validation ; renal ; colon ; TISSUES ; TARGET ; NO ; IDENTIFICATION ; gene expression ; ASSAY ; STABLE EXPRESSION ; POLYMERASE-CHAIN-REACTION ; CHAIN-REACTION ; RIBOSOMAL-RNA ; gastrointestinal stromal tumor ; GASTROINTESTINAL STROMAL TUMORS ; F ; CHAIN ; CELL CARCINOMA ; colon carcinoma ; polymerase chain reaction ; BETA-ACTIN ; clear cell renal cell carcinoma ; equivalence test ; GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE ; housekeeping gene ; HOUSEKEEPING GENES ; INTERNAL STANDARDS ; MESSENGER-RNA LEVELS ; normalization ; quantitative RT-PCR ; REAL-TIME ; reference gene ; REVERSE TRANSCRIPTION ; TIME RT-PCR
    Abstract: In quantitative reverse transcription-polymerase chain reaction (qRT-PCR), normalization using reference genes is a common useful approach, but the validation of suitable reference genes remains a crucial problem. Use of unconfirmed reference genes may lead to misinterpretation of the expression of target genes. The aim of this study was to adapt an adequate statistical approach to identify and validate reference genes suitable for normalization in qRT-PCR assays. We introduce the equivalence test for the identification of stably expressed reference genes. To evaluate the advantages of this test, the expression of five genes widely used as reference genes (18S, B2M, HPRT1, LMNB1, and SDHA), and of two target genes (TP53 and MMP2), was determined with qRT-PCR in different tissues (clear cell renal cell carcinoma, colon carcinoma, and gastrointestinal stromal tumors). We demonstrate that a stable expression of a reference gene in one tumor type does not predict a stable expression in another tumor type. In addition, we found that even within one tumor type, the expression of a reference gene was not stable for different biological groupwise comparisons. These observations confirm that there is no universal reference gene and underline the importance of specific validation of potential reference genes for any experimental condition. (C) 2004 Elsevier Inc. All rights reserved
    Type of Publication: Journal article published
    PubMed ID: 15519565
    Signatur Availability
    BibTip Others were also interested in ...
  • 9
    Keywords: CANCER ; EXPRESSION ; tumor ; AGENTS ; CELL ; QUANTIFICATION ; SUPPORT ; TOOL ; GENE ; GENES ; PATIENT ; DNA ; BREAST ; breast cancer ; BREAST-CANCER ; TARGET ; ASSAY ; PROMOTER ; REPRODUCIBILITY ; EFFICACY ; mass spectrometry ; CELL-LINE ; DNA methylation ; PCR ; MASS-SPECTROMETRY ; POLYMERASE-CHAIN-REACTION ; UNITED-STATES ; sensitivity ; METHYLATION ; AGENT ; polymerase chain reaction ; tumor suppressor gene ; USA ; CLINICAL-RESPONSE ; tumor suppressor ; POLYMERASE ; STATE ; 3 ; therapeutic ; THERAPEUTIC TARGET ; LC-MS/MS ; Liquid chromatography-tandem mass spectrometry ; pharmacodynamic
    Abstract: Promoter hypermethylation-associated tumor suppressor gene (TSG) silencing has been explored as a therapeutic target for hypomethylating agents. Promoter methylation change may serve as a pharmacodynamic endpoint for evaluation of the efficacy of these agents and predict the patient's clinical response. Here a liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay has been developed for quantitative regional DNA methylation analysis using the molar ratio of 5-methyl-2'-deoxycytidine (5mdC) to 2'-deoxycytidine (2dC) in the enzymatic hydrolysate of fully methylated bisulfite-converted polymerase chain reaction (PCR) amplicons as the methylation indicator. The assay can differentiate 5% of promoter methylation level with an intraday precision ranging from 3 to 16% using two TSGs: HIN-1 and RASSF1A. This method was applied to characterize decitabine-induced promoter DNA methylation changes of these two TSGs in a breast cancer MCF-7 cell line. Promoter methylation of these TSGs was found to decrease in a dose-dependent manner. Correspondingly, the expression of these TSGs was enhanced. The sensitivity and reproducibility of the method make it a valuable tool for specific gene methylation analysis that could aid characterization of hypomethylating activity on specific genes by hypomethylating agents in a clinical setting
    Type of Publication: Journal article published
    PubMed ID: 19442645
    Signatur Availability
    BibTip Others were also interested in ...
  • 10
    Keywords: PEPTIDE ; Germany ; TOOL ; DOMAIN ; SEQUENCE ; CRYSTAL-STRUCTURE ; PEPTIDES ; STABILITY ; FRAGMENTS ; PREDICTION ; INITIATION ; CLUSTER ; PROGRAM ; RE ; COILED-COIL ; CONFORMATION ; BETA-SHEET PROTEINS ; BUNDLE PROTEIN ; DE-NOVO DESIGN ; HAIRPIN PEPTIDE ; protein structure ; structure stability ; TRANSITION-STATE
    Abstract: The side chain interaction index (SCII) is a method of calculating the propensity for short-range interactions among side chains within a peptide sequence. Here, it is shown that the SCII values of secondary structure elements that have been shown to fold early and independently cluster separately from those of structures that fold later and/or are dependent on long-range interactions. In addition, the SCII values of engineered peptides that spontaneously adopt a particular desired fold in solution are significantly different from those of engineered peptides that fail to exhibit a stable conformation. Thus, the SCII, as a measure of local structural stability, constitutes a useful tool in folding prediction and in protein/peptide engineering. A program that allows rapid calculation of SCIT values is presented. (c) 2006 Elsevier Inc. All rights reserved
    Type of Publication: Journal article published
    PubMed ID: 16860775
    Signatur Availability
    BibTip Others were also interested in ...
Close ⊗
This website uses cookies and the analysis tool Matomo. More information can be found here...