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  • 1
    Keywords: CANCER ; tumor ; CELL ; Germany ; human ; SYSTEM ; SYSTEMS ; DISEASE ; liver ; PROTEIN ; PROTEINS ; SAMPLE ; SAMPLES ; MOLECULES ; TISSUE ; TISSUES ; BIOLOGY ; MOLECULE ; IDENTIFICATION ; MEMBRANE ; INSTABILITY ; ELECTROPHORESIS ; pancreatic cancer ; SECTIONS ; molecular biology ; pancreas ; PANCREATIC-CANCER ; EXTRACTION ; SEPARATION ; USA ; protein fractionation ; CELL-CELL ; EFFECTOR MOLECULE ; ISLETS ; protein extraction
    Abstract: Proteins are the major class of effector molecules in cellular systems. For the identification of functional differences between normal and diseased tissues, a reliable analysis of their protein content is essential. Reproducible isolation and fractionation of intact proteins are important in this respect, but their complexity in structure and concentration, their close interaction, and their instability represent major challenges. For protein isolation in tissues, the breakdown of cell-cell and cell-matrix connections within a tissue without affecting protein quality is a critical factor. We compared different processes for a compartmental protein preparation from pancreatic tissue, one of the most challenging tissues for protein isolation because of its high protease content. Success of the different procedures varied greatly. Based on a scheme of tissue-slicing and subsequent cell isolation, we established a reliable workflow for the fractional extraction of cytosolic proteins, membrane and organelle proteins, nuclear proteins, and cytoskeletal filaments. The tissue slices also allow for a representative confirmation of individual samples' cellular status by histochemical processes, and a proper separation or mixing of cellular material from across a tumor if required
    Type of Publication: Journal article published
    PubMed ID: 19450236
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  • 2
    Keywords: brain ; CANCER ; CELLS ; ENDOTHELIAL-CELLS ; EXPRESSION ; tumor ; CELL ; Germany ; LUNG-CANCER ; SYSTEM ; DISEASE ; PROTEIN ; TISSUE ; MICE ; PATIENT ; ANTIGEN ; T-CELL ; T-CELLS ; BONE-MARROW ; MEMORY ; RECOGNITION ; MOUSE ; IDENTIFICATION ; LYMPHOMA ; EFFICACY ; MELANOMA ; MASS-SPECTROMETRY ; HEAD ; NECK ; EPITOPE ; IMMUNOTHERAPY ; IMMUNOGENICITY ; CANCER PATIENTS ; CALCIUM-BINDING PROTEINS ; TUMOR-ASSOCIATED ANTIGENS ; NECK-CANCER ; brain tumor ; head and neck cancer ; endothelial cells ; proteome ; EGFR ; SEPARATION ; EXPRESSION PROFILES ; Type ; HEAD-AND-NECK
    Abstract: Identifying the antigens that have the potential to trigger endogenous antitumor responses in an individual cancer patient is likely to enhance the efficacy of cancer immunotherapy, but current methodologies do not efficiently identify such antigens. This study describes what we believe to be a new method of comprehensively identifying candidate tissue antigens that spontaneously cause T cell responses in disease situations. We used the newly developed automated, two-dimensional chromatography system PF2D to fractionate the proteome of human tumor tissues and tested protein fractions for recognition by preexisting tumor-specific CD4(+) Th cells and CTLs. Applying this method using mice transgenic for a TCR that recognizes an OVA peptide presented by MHC class I, we demonstrated efficient separation, processing, and cross-presentation to CD8(+) T cells by DCs of OVA expressed by the OVA-transfected mouse lymphoma RMA-OVA. Applying this method to human tumor tissues, we identified MUC1 and EGFR as tumor-associated antigens selectively recognized by T cells in patients with head and neck cancer. Finally, in an exemplary patient with a malignant brain tumor, we detected CD4(+) and CD8(+) T cell responses against two novel antigens, transthyretin and calgranulin B/S100A9, which were expressed in tumor and endothelial cells. The immunogenicity of these antigens was confirmed in 4 of 10 other brain tumor patients. This fast and inexpensive method therefore appears suitable for identifying candidate T cell antigens in various disease situations, such as autoimmune and malignant diseases, without being restricted to expression by a certain cell type or HLA allele
    Type of Publication: Journal article published
    PubMed ID: 20458140
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  • 3
    Keywords: CANCER ; EXPRESSION ; QUANTIFICATION ; IDENTIFICATION ; AMINO-ACIDS ; POSTTRANSCRIPTIONAL REGULATION ; MESSENGER-RNAS ; BIOGENESIS ; TRANSLATION INITIATION ; CELL-CULTURE ; MICRORNA-BINDING-SITES
    Abstract: Background: MicroRNAs are 22 nucleotides long non-coding RNAs and exert their function either by transcriptional or translational inhibition. Although many microRNA profiles in different tissues and disease states have already been discovered, only little is known about their target proteins. The microRNA miR-155 is deregulated in many diseases, including cancer, where it might function as an oncoMir. Methodology/Principal Findings: We employed a proteomics technique called "stable isotope labelling by amino acids in cell culture" (SILAC) allowing relative quantification to reliably identify target proteins of miR-155. Using SILAC, we identified 46 putative miR-155 target proteins, some of which were previously reported. With luciferase reporter assays, CKAP5 was confirmed as a new target of miR-155. Functional annotation of miR-155 target proteins pointed to a role in cell cycle regulation. Conclusions/Significance: To the best of our knowledge we have investigated for the first time miR-155 target proteins in the HEK293T cell line in large scale. In addition, by comparing our results to previously identified miR-155 target proteins in other cell lines, we provided further evidence for the cell line specificity of microRNAs
    Type of Publication: Journal article published
    PubMed ID: 21799781
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  • 4
    Keywords: CANCER ; EXPRESSION ; INHIBITOR ; tumor ; carcinoma ; CELL ; Germany ; CLASSIFICATION ; DIAGNOSIS ; RISK ; PROTEIN ; SAMPLES ; PATIENT ; BIOLOGY ; ASSOCIATION ; FIELD ; ALPHA ; STAGE ; IDENTIFICATION ; IN-SITU ; immunohistochemistry ; genetics ; ABERRATIONS ; MASS-SPECTROMETRY ; ONCOGENE ; HUMAN-PAPILLOMAVIRUS ; SQUAMOUS-CELL CARCINOMA ; HEAD ; NECK ; RISK ASSESSMENT ; heredity ; BIOPSY ; protein expression ; PROTEOMICS ; PROTEOMIC ANALYSIS ; NECK-CANCER ; CELL CARCINOMA ; ONCOLOGY ; TUMORIGENESIS ; ARRAY ; HNSCC ; LYMPH-NODE METASTASIS ; development ; analysis ; PROFILES ; EVENTS ; protein biomarkers ; HISTOLOGY ; FRAGMENT ; SELDI-TOF-MS ; BIOPSIES ; CLINICAL-IMPLICATIONS ; aberration ; comparison ; acyl-CoA-binding protein ; CANCERIZATION ; field cancerization ; GENETICALLY ALTERED FIELDS ; HUMAN NEUTROPHIL PEPTIDE-1 ; TUMOR-DISTANT EPITHELIA
    Abstract: Development of head and necksquamous cell carcinoma (HNSCC) is a multistep process and in many cases involves a phenomenon coined 'field cancerization'. In order to identify changes in protein expression occurring at different stages of tumorigenesis and field cancerization, we analysed 113 HNSCCs and 73 healthy, 99 tumor-distant and 18 tumor-adjacent squamous mucosae by SELDI-TOF-MS on IMAC30 ProteinChip Arrays. Forty-eight protein peaks were differentially expressed between healthy mucosa and HNSCC. Calgizarrin (S100A11), the Cystein proteinase inhibitor Cystatin A, Acyl-CoA-binding protein, Stratifin (14-3-3 sigma), Histone H4, alpha- and beta-Hemoglobin, a C-terminal fragment of beta-hemoglobin and the alpha-defensins 1-3 were identified by mass spectrometry. The alpha-defensins showed various alterations in expression as validated by immunohistochemistry (IHC). Supervised prediction analysis revealed excellent classification of healthy mucosa (94.5% correctly classified) and tumor samples (92.9% correctly classified). Application of this classifier to the tumor-adjacent and tumor-distant mucosa samples disclosed dramatic changes: only 59.6% of the tumor-distant biopsies were classified as normal, 27.3% were predicted as aberrant or HNSCC. Strikingly, 72% of the tumor-adjacent mucosae were predicted as aberrant. These data provide evidence for the existence of genetically altered fields with inconspicuous histology. Comparison of the protein profiles in the tumor-distant-samples with clinical outcome of 32 patients revealed a significant association between aberrant profiles with tumor relapse events (P = 0.018; Fisher's exact test, two-tailed). We conclude that proteomic pro. ling in conjunction with protein identification greatly outperforms histopathological diagnosis and may have significant predictive power for clinical outcome and personalized risk assessment
    Type of Publication: Journal article published
    PubMed ID: 16819514
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