Biomedical subjects

Elisabeth Kremmer

Publications and source records attributed to Elisabeth Kremmer.

66 records · Page 4Linked to original sources

PubMed2003

Attenuation of cell adhesion in lymphocytes is regulated by CYTIP, a protein which mediates signal complex sequestration.

An important theme in molecular cell biology is the regulation of protein recruitment to the plasma membrane. Fundamental biological processes such as proliferation, differentiation or leukocyte functions are initiated and controlled through the reversible binding of signaling proteins to phosphorylated membrane components. This is mediated by specialized interaction modules, such as SH2 and PH domains. Cytohesin-1 is an intracellular guanine nucleotide exchange factor, which regulates leukocyte adhesion. The activity of cytohesin-1 is controlled by phospho inositide-dependent membrane recruitment. An interacting protein was identified, the expression of which is upregulated by cytokines in hematopoietic cells. This molecule, CYTIP, is also recruited to the cell cortex by integrin signaling via its PDZ domain. However, stimulation of Jurkat cells with phorbol ester results in re-localization of CYTIP to the cytoplasm, and membrane detachment of cytohesin-1 strictly requires co-expression of CYTIP. Consequently, stimulated adhesion of Jurkat cells to intracellular adhesion molecule-1 is repressed by CYTIP. These findings outline a novel mechanism of signal chain abrogation through sequestration of a limiting component by specific protein-protein interactions.

Adenosine Triphosphate↗

PubMed2003

Human replication protein A. The C-terminal RPA70 and the central RPA32 domains are involved in the interactions with the 3'-end of a primer-template DNA.

Although the mechanical aspects of the single-stranded DNA (ssDNA) binding activity of human replication protein A (RPA) have been extensively studied, only limited information is available about its interaction with other physiologically relevant DNA structures. RPA interacts with partial DNA duplexes that resemble DNA intermediates found in the processes of DNA replication and DNA repair. Limited proteolysis of RPA showed that RPA associated with ssDNA is less protected against proteases than RPA bound to a partial duplex DNA containing a 5'-protruding tail that had the same length as the ssDNA. Modification of both the 70- and 32-kDa subunits, RPA70 and RPA32, respectively, by photoaffinity labeling indicates that RPA can bind the primer-template junction of partial duplex DNAs by interacting with the 3'-end of the primer. The identification of the protein domains modified by the photoreactive 3'-end of the primer showed that domains located in the central part of the RPA32 subunit (amino acids 39-180) and the C-terminal part of the RPA70 subunit (amino acids 432-616) are involved in these interactions.

3' Flanking Region↗

PubMed2003

Significance of individual amino acid residues for coenzyme and substrate specificity of 17beta-hydroxysteroid dehydrogenase from the fungus Cochliobolus lunatus.

17beta-Hydroxysteroid dehydrogenase from the fungus Cochliobolus lunatus (17beta-HSDcl) is a NADPH dependent member of the short-chain dehydrogenase reductase (SDR) superfamily. Recently, we prepared a homology-built structural model of 17beta-HSDcl using the known three-dimensional structure of homologous 1,3,8-trihydroxynaphthalene reductase from the fungus Magnaporthe grisea. This model structure directed our studies of structure-function relationship of the fungal 17beta-HSD, as one of the model enzymes of the SDR superfamily. In this work, we investigated the significance of individual amino acid residues for coenzyme and substrate specificity. We performed site directed mutagenesis of R28, a basic residue conserved in most NADPH dependent SDR structures; T200, found only in Streptomyces hydrogenans 3alpha,20beta-HSD and Drosophila alcohol dehydrogenases; and H230, a residue corresponding to the substrate specificity important H221 in human 17beta-HSD type 1. All recombinant proteins were expressed in Escherichia coli and purified to homogeneity. Kinetic evaluation of individual mutations was performed by analysis of progress curves of interconversions between 4-estrene-3,17-dione and 4-estrene-17beta-ol-3-one, in the presence of NADPH and NADP(+); according to the Theorell-Chance reaction mechanism. The results demonstrate the role of the selected amino acid residues; R28 seems to interact with the NADPH 2'-phosphate group; T200 may be involved in binding and dissociation of NADPH/NADP(+); while H230 and the neighboring A231 appears not to be responsible for substrate specificity of 17beta-HSDcl.

17-Hydroxysteroid Dehydrogenases↗

PubMed2003

HCN channels are expressed differentially in retinal bipolar cells and concentrated at synaptic terminals.

Hyperpolarization-activated and cyclic nucleotide-gated (HCN) channels codetermine the integrative behaviour of neurons and shape their response to synaptic stimulation. We used immunohistochemistry and patch-clamp recording to study the composition and distribution of HCN channels in the rat retina. All four HCN channel isoforms (HCN1-4) are expressed differentially in the retina. In particular, different classes of bipolar cells have a different inventory of HCN channels. We found no evidence for the formation of heterooligomeric HCN channels. HCN channels are densely clustered at synaptic terminals of bipolar cells and photoreceptors. This suggests that HCN channels are involved in the control of transmitter release.

Animals↗

PubMed2002

Subunit stoichiometry of the CNG channel of rod photoreceptors.

Cyclic nucleotide-gated (CNG) channels play a central role in the conversion of sensory stimuli into electrical signals. CNG channels form heterooligomeric complexes built of A and B subunits. Here, we study the subunit stoichiometry of the native rod CNG channel by chemical crosslinking. The apparent molecular weight (M(w)) of each crosslink product was determined by SDS-PAGE, and its composition was analyzed by Western blotting using antibodies specific for the A1 or B1 subunit. The number of crosslink products and their M(w) as well as the immunological identification of A1 and B1 subunits in the crosslink products led us to conclude that the native rod CNG channel is a tetramer composed of three A1 and one B1 subunit. This is an example of violation of symmetry in tetrameric channels.

Animals↗

PubMed2002

Tumor-derived heat shock protein 70 peptide complexes are cross-presented by human dendritic cells.

Our study demonstrates that tumor-derived heat shock protein (HSP)70 chaperones a tyrosinase peptide and mediates its transfer to human immature dendritic cells (DCs) by receptor-dependent uptake. Human tumor-derived HSP70 peptide complexes (HSP70-PC) thus have the immunogenic potential to instruct DCs to cross-present endogenously expressed, nonmutated, and tumor antigenic peptides that are shared among tumors of the melanocytic lineage for T cell recognition. T cell stimulation by HSP70-instructed DCs is dependent on the Ag bound to HSP70 in that only DCs incubated with HSP70-PC purified from tyrosinase-positive (HSP70-PC/tyr(+)) but not from tyrosinase-negative (HSP70-PC/tyr(-)) melanoma cells resulted in the specific activation of the HLA-A*0201-restricted tyrosinase peptide-specific cytotoxic T cell clone. HSP70-PC-mediated T cell stimulation is very efficient, delivering the tyrosinase peptide at concentrations as low as 30 ng/ml of HSP70-PC for T cell recognition. Receptor-dependent binding of HSP70-PC and active cell metabolism are prerequisites for MHC class I-restricted cross-presentation and T cell stimulation. T cell stimulation does not require external DC maturation signals (e.g., exogenously added TNF-alpha), suggesting that signaling DC maturation is an intrinsic property of the HSP70-PC itself and related to receptor-mediated binding. The cross-presentation of a shared human tumor Ag together with the exquisite efficacy are important new aspects for HSP70-based immunotherapy in clinical anti-cancer vaccination strategies, and suggest a potential extension of HSP70-based vaccination protocols from a patient-individual treatment modality to its use in an allogeneic setting.

Antigen Presentation↗

PubMed2002

Histone methylation by the Drosophila epigenetic transcriptional regulator Ash1.

The establishment and maintenance of mitotic and meiotic stable (epigenetic) transcription patterns is fundamental for cell determination and function. Epigenetic regulation of transcription is mediated by epigenetic activators and repressors, and may require the establishment, 'spreading' and maintenance of epigenetic signals. Although these signals remain unclear, it has been proposed that chromatin structure and consequently post-translational modification of histones may have an important role in epigenetic gene expression. Here we show that the epigenetic activator Ash1 (ref. 5) is a multi-catalytic histone methyl-transferase (HMTase) that methylates lysine residues 4 and 9 in H3 and 20 in H4. Transcriptional activation by Ash1 coincides with methylation of these three lysine residues at the promoter of Ash1 target genes. The methylation pattern placed by Ash1 may serve as a binding surface for a chromatin remodelling complex containing the epigenetic activator Brahma (Brm), an ATPase, and inhibits the interaction of epigenetic repressors with chromatin. Chromatin immunoprecipitation indicates that epigenetic activation of Ultrabithorax transcription in Drosophila coincides with trivalent methylation by Ash1 and recruitment of Brm. Thus, histone methylation by Ash1 may provide a specific signal for the establishment of epigenetic, active transcription patterns.

Animals↗

PubMed2002

Expression of viral and human dUTPase in Epstein-Barr virus-associated diseases.

Deoxyuridine triphosphatase (dUTPase) catalyses the hydrolysis of dUTP to dUMP and pyrophosphate thus preventing the incorporation of uracil into replicating DNA. Previous studies of several virus models have suggested that viral dUTPases may be required for virus replication in resting cells whereas in proliferating cells cellular dUTPase may substitute for a mutant viral protein. Using monoclonal antibodies and immunohistochemistry, Epstein-Barr virus-associated non-neoplastic and neoplastic diseases were studied for the expression of viral and human dUTPases. Oral hairy leukoplakia, an AIDS-associated lesion of the tongue, is known to support EBV replication in the upper epithelial cell layers. In agreement with this, strong focal expression of EBV dUTPase was detected in the upper epithelial cell layers of oral hairy leukoplakia whereas expression of human dUTPase was confined to the basal proliferative cell compartment. Furthermore, in infectious mononucleosis tonsils, rare scattered small lymphoid cells expressed EBV dUTPase, consistent with the expression pattern of other EBV lytic cycle antigens. These findings are in agreement with the notion that EBV replicates in resting cells. Three EBV-associated tumours, Hodgkin lymphoma, Burkitt lymphoma and nasopharyngeal carcinoma, lacked detectable expression of EBV dUTPase, in agreement with the notion that EBV infection is largely latent in these tumours. By contrast, expression of human dUTPase was observed regularly in these tumours. These results suggest that EBV dUTPase may be a suitable target for anti-viral therapy and that inhibitors of human dUTPase should prove useful for the treatment of human tumours, including EBV-associated cancers.

Antibodies, Monoclonal↗

PubMed2002

Progressive loss of PAX9 expression correlates with increasing malignancy of dysplastic and cancerous epithelium of the human oesophagus.

Pax genes encode a family of transcription factors that play key roles in embryonic development. Whereas the functions of Pax genes in the adult organism are largely unknown, upregulated Pax gene expression has been implicated in tumourigenesis. In this study, PAX9-specific monoclonal antibodies have been generated and it has been shown that PAX9 protein is expressed in the normal epithelium of the adult human oesophagus. PAX9 expression was either lost or significantly reduced in the majority of invasive carcinomas and epithelial dysplasias, the latter representing precancerous lesions. Notably, the percentage of PAX9-positive cells within the epithelium decreased with increasing malignancy of the epithelial lesion. These results identify PAX9 as a sensitive marker for deregulated differentiation of oesophageal keratinocytes and indicate a role for PAX9 in the normal differentiation process of internal stratified squamous epithelia. These data suggest that upregulated PAX9 expression is not required for the formation of the majority of squamous cell carcinomas of the human oesophagus.

Biomarkers↗

PubMed2002

Functional allelic loss detected at the protein level in archival human tumours using allele-specific E-cadherin monoclonal antibodies.

Immunohistochemical analysis has been used to show that expression of the homophilic cell-to-cell adhesion molecule, E-cadherin, is frequently altered in human cancers, including gastric and breast carcinoma. Besides genetic down-regulation, structural mutations such as in-frame deletions of exon 8 and exon 9 were frequently found; these may affect the binding of monoclonal antibodies used for immunohistochemical analysis. In this study it was found that antibodies HECD-1 and E9, two monoclonal antibodies often used in E-cadherin immunoanalysis, react with epitopes present at least in part in exon 8 and exon 9, respectively. This study generated and characterized a mutation-specific monoclonal antibody, E-cad delta 8-1, reacting with the mutant protein lacking exon 8 but not with the wild-type molecule. By using E-cad delta 8-1 and HECD-1, it was possible separately to analyse the immunoreactivity of mutant and normal E-cadherin proteins, respectively, in an allele-specific manner in archival material. A similar analysis was performed using E9 and the previously characterized mutation-specific antibody E-cad delta 9-1. Typically, in gastric and breast cancer harbouring E-cadherin splice site gene mutations, the mutant proteins were expressed but the wild-type protein was not detected in malignant tissues. These results indicate that variant-specific monoclonal antibodies can be used to identify differentially expressed E-cadherin proteins. For immunohistochemical analysis of E-cadherin, at least two different monoclonal antibodies should be used to exclude alterations of the epitopes resulting in failure to detect a mutant protein.

Antibodies, Monoclonal↗

PubMed2002

Overexpression, purification, and biochemical characterization of the extracellular human CD83 domain and generation of monoclonal antibodies.

CD83 is a 45-kDa glycoprotein and member of the immunoglobulin (Ig) superfamily. It is the best known marker for mature dendritic cells. Although the precise function of CD83 is not known, its selective expression and upregulation together with the costimulators CD80 and CD86 suggests an important role of CD83 in the induction of immune responses. To perform functional studies and to elucidate its mode of action it is vital to obtain recombinant expressed and highly purified CD83 molecules. Therefore, the external Ig domain of human CD83 (hCD83ext) was expressed as a GST fusion protein (GST-hCD83ext) and the soluble protein was purified under native conditions. The fusion protein was purified using GSTrap columns followed by anion-exchange chromatography. GST-hCD83ext was then cleaved using thrombin and soluble hCD83ext was further purified using GSTrap columns and finally by a preparative gel filtration as a polishing step and used for further characterization. The purified GST-hCD83 fusion protein was also used to generate monoclonal anti-CD83 antibodies in a rat system. Two different monoclonal antibodies were generated. Using these antibodies, CD83 was specifically recognized in FACS and Western blot analyses. Furthermore, we showed that native CD83 is glycosylated and that this glycosylation influences the binding of the antibodies in Western blot analyses. Finally, the purified hCD83ext protein was analyzed by one-dimensional NMR and these analyses strongly indicate that hCD83ext is folded and could therefore be used for further structural and functional studies.

Animals↗

PubMed2002

Guanylate-binding protein-1 expression is selectively induced by inflammatory cytokines and is an activation marker of endothelial cells during inflammatory diseases.

During angiogenesis and inflammatory processes, endothelial cells acquire different activation phenotypes, whose identification may help in understanding the complex network of angiogenic and inflammatory interactions in vivo. To this goal we investigated the expression of the human guanylate-binding protein (GBP)-1 that is highly induced by inflammatory cytokines (ICs) and, therefore, may characterize IC-activated cells. Using a new rat monoclonal antibody raised against GBP-1, we show that GBP-1 is a cytoplasmic protein and that its expression in endothelial cells is selectively induced by interferon-gamma, interleukin-1alpha, interleukin-1beta, or tumor necrosis factor-alpha, but not by other cytokines, chemokines, or growth factors. Moreover, we found that GBP-1 expression is highly associated with vascular endothelial cells as confirmed by the simultaneous detection of GBP-1 and the endothelial cell-associated marker CD31 in a broad range of human tissues. Notably, GBP-1 expression was undetectable in the skin, but it was highly induced in vessels of skin diseases with a high-inflammatory component including psoriasis, adverse drug reactions, and Kaposi's sarcoma. These results indicate that GBP-1 is a novel cellular activation marker that characterizes the IC-activated phenotype of endothelial cells.

Biomarkers↗