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R Schäfer

Publications and source records attributed to R Schäfer.

At least 19 recordsLinked to original sources
PubMed2006

Oncogenic HRAS suppresses clusterin expression through promoter hypermethylation.

Silencing of gene expression by methylation of CpG islands in regulatory elements is frequently observed in cancer. However, an influence of the most common oncogenic signalling pathways onto DNA methylation has not yet been investigated thoroughly. To address this issue, we identified genes suppressed in HRAS-transformed rat fibroblasts but upregulated after treatment with the demethylating agent 5-Aza-2-deoxycytidine and with the MEK1,2 inhibitor U0126. Analysis of gene expression by microarray and Northern blot analysis revealed the MEK/ERK target genes clusterin, matrix metalloproteinase 2 (Mmp2), peptidylpropyl isomerase C-associated protein, syndecan 4, Timp2 and Thbs1 to be repressed in the HRAS-transformed FE-8 cells in a MEK/ERK- and methylation-dependent manner. Hypermethylation of putative regulatory elements in HRAS-transformed cells as compared to immortalized fibroblasts was detected within a CpG island 14.5 kb upstream of clusterin, within the clusterin promoter and within a CpG island of the Mmp2 promoter by bisulphite sequencing. Furthermore, hypermethylation of the clusterin promoter was observed 10 days after induction of HRAS in immortalized rat fibroblasts and a clear correlation between reduced clusterin expression and hypermethlyation could also be observed in distinct rat tissues. These results suggest that silencing of individual genes by DNA methylation is controlled by oncogenic signalling pathways, yet the mechanisms responsible for initial target gene suppression are variable.

Animals↗
PubMed2006

The PI3K inhibitor LY294002 blocks drug export from resistant colon carcinoma cells overexpressing MRP1.

Multidrug resistance may be achieved by the activation of membrane transporters, detoxification, alterations in DNA repair or failure in apoptotic pathways. Recent data have suggested an involvement of mitogenic signalling pathways mediated by Ras and phosphoinositol-3-kinase (PI3K/Akt) in controlling multidrug resistance. Since these pathways are important targets for therapeutic interference, we sought to investigate whether blocking effectors kinases by specific inhibitors would result in a sensitization toward cytotoxic drugs. We found that cotreatment of drug-resistant HT29RDB colon cancer cells with the topoisomerase inhibitor doxorubicin and the PI3K-inhibitor LY294002 resulted in massive apoptosis, while cotreatment with the Mek inhibitors PD98059 or U0126 had no effect. This suggested that the PI3K-pathways controls cell survival and drug resistance in these cells. Besides blocking Akt phosphorylation, the PI3K-inibitor increased the intracellular doxorubicin concentration threefold. LY294002 inhibits drug export in a competitive manner as revealed by measuring drug efflux in the presence and the absence of inhibitor. The efficacy of drug efflux inhibition by LY294002 was similar to that achieved by the MRP1 inhibitors MK571 and genistein. We conclude that the PI3K inhibitor LY294002 may have therapeutic potential when combined with doxorubicin in the treatment of MRP1-mediated drug resistance.

Antibiotics, Antineoplastic↗
PubMed2005

Experimental verification of fidelity decay: from perturbative to fermi golden rule regime.

The scattering matrix was measured for a flat microwave cavity with classically chaotic dynamics. The system can be perturbed by small changes of the geometry. We define the "scattering fidelity" in terms of parametric correlation functions of scattering matrix elements. In chaotic systems and for weak coupling, the scattering fidelity approaches the fidelity of the closed system. Without free parameters, the experimental results agree with random matrix theory in a wide range of perturbation strengths, reaching from the perturbative to the Fermi golden rule regime.

Journal Article↗
PubMed2005

Endocytosis mechanism of P2Y2 nucleotide receptor tagged with green fluorescent protein: clathrin and actin cytoskeleton dependence.

Extracellular nucleotides exert a large number of physiological effects through activation of P2Y receptors. We expressed rat P2Y2 (rP2Y2) receptor, tagged with green fluorescent protein (GFP) in HEK-293 cells and visualized receptor translocation in live cells by confocal microscopy. Functional receptor expression was confirmed by determining [Ca2+]i responses. Agonist stimulation caused a time-dependent translocation of the receptor from the plasma membrane to the cytoplasm. Rearrangement of the actin cytoskeleton was observed during agonist-mediated rP2Y2-GFP receptor internalization. Colocalization of the internalized receptor with early endosomes, clathrin and lysosomes was detected by confocal microscopy. The inhibition of receptor endocytosis by either high-density medium or chlorpromazine in the presence of UTP indicates that the receptor was internalized by the clathrin-mediated pathway. The caveolin-mediated pathway was not involved. Targeting of the receptor from endosomes to lysosomes seems to involve the proteasome pathway, because proteasomal inhibition increased receptor recycling back to the plasma membrane.

Actins↗
PubMed2004

The ontogeny of soleus muscles in mdx and wild type mice.

The satellite cell, the organotypic muscle stem cell, is the key element in ontogenetic and load induced muscle fibre growth and repair. It is therefore possible that the satellite pool becomes exhausted with age, especially in mdx mice where dystrophin deficiency results in skeletal muscle degeneration. We compared structural criteria and satellite cell frequencies in soleus muscles of 26 mdx and 23 wild type mice aged between 26 and 720 days. The total number of muscle fibres was similar in both groups and remained stable throughout life, except for an early increase in wild type mice. However, in mdx muscles there was always a proportion of small-diameter fibres which resulted in a reduction in the effective myogenic area on cross-section, whereas total cross-sectional area and muscle weights were increased relative to controls throughout life. In adult animals, the frequency and numbers of satellite cells remained stable with age and were similar in both animal groups. Satellite cell numbers showed some considerable variation between individual animals, although with a markedly smaller variability between results of the same animal, pointing to the satellite cell pool being an individual variant.

Aging↗
PubMed2004

[Assessment and outcome of perinatal and neonatal care in North Rhine area].

The objective of the quality assurance program "Perinatalerhebung" and "Neonatalerhebung" is to ameliorate the perinatal and neonatal care in German hospitals. Standardised data defined by a team of experts in the early seventies are documented for more than 20 years in the North Rhine area. The documentation of more than 1.6 million deliveries created a data pool that served as a thesaurus for the improvement of medical care in obstetrical units. The same has been achieved for neonatal care on the documentation basis of more than 168,000 newborn infants admitted to a neonatal department. The results show that the approach to evaluate continuously the data monitored enables improvement in care. The support given is the anonymous collection of the documentation sheets/data sets and their evaluation by an expert committee. The results show that obstetrical management changed and the measurable results improved demonstrably. The efforts seem to be worthwhile in view of the fact that less than 1 % of the total cost of care has been spent for this project.

Female↗
PubMed2004

Hepatoid thymic carcinoma: report of a case.

We describe the clinicopathologic findings in a so far unrecognized thymic tumor. The tumor occurred in a 70-year-old woman with respiratory distress but neither myasthenia gravis nor other symptoms. Metastases or another primary tumor were absent. The well-circumscribed neoplasm was located in the thymic region, measured 18 x 12 x 8 cm, and showed a homogeneous, tan-colored, soft cut surface. By histology, the tumor lacked a true capsule and a lobular growth pattern, was almost devoid of stroma, and infiltrated among remnant thymus lobules. The polygonal tumor cells formed solid sheets, trabeculae, or occurred as single cells that resembled hepatocytes. Proliferative activity was low. Portal structures, sinuses, and bile were absent as were areas of conventional thymoma, adenocarcinoma, or germ cell tumor. The tumor expressed cytokeratins 7 and 19, alpha1-antitrypsin, alpha1-antichymotrypsin, and hep-Par-1. Alpha-fetoprotein (AFP), human beta-chorionic gonadotropin (beta-HCG), placental alkaline phosphatase, CD5, CD30, CD31, CD34, CD45, CD68, CD99, S-100, HMB45, desmin, actin, or neuroendocrine markers were not expressed, and intratumorous CD1a+ or TdT+ immature T cells were absent. AFP was repeatedly undetectable in the blood. Mediastinal tumor recurrence was detected 6 months after surgery. Following radiochemotherapy, the patient has remained free of disease for 26 months. We conclude that this tumor is a thymic carcinoma (WHO type C thymoma). A diagnosis of hepatoid yolk sack tumor appears unlikely considering absence of a bona fide germ cell component, lack of AFP expression, and the patient's female gender. Because of its morphologic and immunohistochemical features, we propose the term "hepatoid thymic carcinoma" for this new type of thymic carcinoma.

Aged↗
PubMed2004

Spill-over of European bat lyssavirus type 1 into a stone marten (Martes foina) in Germany.

European bat lyssavirus type 1 (EBLV-1, genotype 5) is known to endemically circulate in insectivorous bat populations in Germany. In August 2001, a rabies suspect stone marten (Martes foina) was found in the city of Burg (Saxony-Anhalt, Germany) and was sent to the regional veterinary laboratory for routine rabies diagnosis. Whereas brain samples repeatedly tested negative in the fluorescent antibody test for classical rabies virus (genotype 1), the mouse inoculation test and the rabies tissue culture inoculation test yielded positive results. Rabies viral RNA was also detected in the stone marten brain sample both by nested and heminested RT-PCR specific for the nucleoprotein gene and for the nucleoprotein phosphoprotein junction of rabies virus. The amplification products were sequenced to genotype the isolate. Sequence data obtained from the first-round RT-PCR products were analysed and the suspect stone marten isolate was confirmed as a rabies related virus (EBLV-1a). Phylogenetic comparison with sequences from recent genotype five isolates from Germany and Denmark showed that it was closely related to a previous isolate of EBLV-1 from a serotine bat in Saxony-Anhalt obtained in the same year in an area adjacent to the place where the EBLV-1 infected stone marten was found. Both EBLV-1 isolates share a 99.5% identity. This is the first report of an EBLV-1a spill-over from an insectivorous bat into wildlife in Europe.

Animals↗
PubMed2002

HERG K(+) currents in human prolactin-secreting adenoma cells.

To investigate the presence and possible function of ether-à-go-go-related gene (erg) K(+) channels in human lactotroph cells (HERG channels), primary cultures were prepared from human prolactinoma tissue. In almost all primary cultures, HERG currents could be recorded in identified prolactin cells using an external high-K(+) solution. The antiarrhythmic agent E-4031, a specific blocker of erg channels, served to isolate HERG currents as the drug-sensitive currents. In cells of two tumours tested, thyrotropin-releasing hormone significantly reduced the amplitude of the HERG currents. The potential dependence of HERG current availability and the deactivation kinetics differed significantly even between prolactin cells derived from one adenoma. For comparison, corresponding values were obtained for heterologously expressed rat erg1, erg2 and erg3 channels. The expression of the three HERG channel subunits was investigated in nine human adenomas using RT-PCR. Transcripts for HERG1 were present in all adenomas and although transcripts for HERG2 and HERG3 were also detected, their expression level was more variable. The results demonstrate the functional expression of HERG channels in human prolactin-secreting tumours and are compatible with a physiological role for these channels in the control of prolactin secretion, as has been shown in normal rat lactotroph cells.

Animals↗
PubMed2002

Transition from Gaussian-orthogonal to Gaussian-unitary ensemble in a microwave billiard with threefold symmetry.

Recently it has been shown that time-reversal invariant systems with discrete symmetries may display, in certain irreducible subspaces, spectral statistics corresponding to the Gaussian-unitary ensemble (GUE) rather than to the expected orthogonal one (GOE). A Kramers-type degeneracy is predicted in such situations. We present results for a microwave billiard with a threefold rotational symmetry and with the option to display or break a reflection symmetry. This allows us to observe the change from GOE to GUE statistics for one subset of levels. Since it was not possible to separate the three subspectra reliably, the number variances for the superimposed spectra were studied. The experimental results are compared with a theoretical and numerical study considering the effects of level splitting and level loss.

Journal Article↗
PubMed2002

Formation of noncanonical high molecular weight caspase-3 and -6 complexes and activation of caspase-12 during serum starvation induced apoptosis in AKR-2B mouse fibroblasts.

Apoptosis is mainly brought about by the activation of caspases, a protease family with unique substrate selectivity. In mammals, different complexes like the DISC complex or the apoptosome complexes have been delineated leading to the cleavage and thus activation of the executioner caspases. Although caspase-3 is the main executioner caspase in apoptosis induced by serum starvation in AKR-2B fibroblasts as demonstrated by affinity labeling with YVK(-bio)D.aomk and partial purification of cytosolic extracts by high performance ion exchange chromatography, its activation is apparently caused by a noncanonical pathway: (1) Expression of CrmA, an inhibitor of caspase-8, failed to suppress apoptosis; (2) There was no formation of high molecular weight complexes of Apaf-1 indicative for its activation. Furthermore no cleavage of caspase-9 was observed. But surprisingly, gelfiltration experiments revealed the distribution of caspase-3 and -6 into differently sized high molecular weight complexes during apoptosis. Though the apparent molecular weights of the complexes containing caspase-3 (600 kD for apoptosome and 250 kD for microapoptosome) are in accordance with recently published data, the activity profiles differ strikingly. In AKR-2B cells caspase-3 is mainly recovered as uncomplexed enzyme and in much lower levels in the apoptosomes. Remarkably, the 600 kD and 250 kD complexes containing activated caspase-3 were devoid of Apaf-1 and cytochrome c. In addition a new 450 kD complex containing activated caspase-6 was found that is clearly separated from the caspase-3 containing complexes. Furthermore, we disclose for the first time the activation of caspase-12 in response to serum starvation. Activated caspase-12 is detectable as non-complexed free enzyme in the cytosol.

Animals↗
PubMed2001

Impact of cardiac transplantation on molecular pathology of ET-1, VEGF-C, and mitochondrial metabolism and morphology in dilated versus ischemic cardiomyopathic patients.

Little is known about the long-term impact of cardiac transplantation on activity and modifications of endothelin (ET)-1 system, vascular endothelial growth factor (VEGF), and mitochondrial metabolism and morphology in patients with ischemic cardiomyopathy (ICM) versus dilated cardiomyopathy (DCM). Messenger RNA (mRNA) expression levels of ET-1, endothelin converting enzyme (ECE)-1, VEGF-C, carnitine palmitoyltransferase (CPT)-1, and carnitine acetyltransferase (CARAT), as well as the number of normal, edematous, and degenerated mitochondria were assessed in left ventricular biopsies of 21 patients with DCM and 20 with ICM (New York Heart Association class III-IV) before and up to 3 months after cardiac transplantation. Cardiac samples of donated, nonfailing hearts served as controls (n=10). In cardiac biopsies of both ICM and DCM patients, ET-1, VEGF-C, CPT-1, and CARAT mRNA were up-regulated, whereas ECE-1 mRNA was down-regulated (P<0.05). Degenerated mitochondria had the highest number in both groups, followed by normal and edematous mitochondria. After cardiac transplantation, in ICM patients impaired gene expression levels decreased to, or below, normal levels, and the number of normal mitochondria increased (P<0.05). In implanted hearts of DCM patients, however, up-regulated ET-1 transcript levels persisted and the number of normal mitochondria decreased, whereas the number of degenerated mitochondria increased (P<0.05), and edematous mitochondria remained unchanged in number. These results show that cardiac transplantation corrects the impaired hemodynamic and echocardiographic parameters in both groups, whereas in DCM, the molecular pathology of ET-1 system and mitochondria persists. Therefore, it is more likely that these changes are the cause rather than a consequence of DCM.

Adult↗
PubMed2001

Selective degradation of the PKC-epsilon isoform during cell death in AKR-2B fibroblasts.

The protein kinase C (PKC) family of serine/threonine protein kinases is involved in intracellular signals that regulate growth, differentiation, and apoptosis. AKR-2B cells express the PKC isoforms alpha, gamma, epsilon, lambda, mu, und zeta (J. Hoppe, R. Schäfer, V. Hoppe, and A. Sachinidis, Cell Death Differ. 6, 546-556). Here we show that during serum starvation only PKC-epsilon was cleaved. An N-terminal fragment of 42 kDa remained associated with subcellular components, presumably the Golgi apparatus. The C-terminal part (catalytic domain) was further degraded and was no longer detectable in vivo. As published before, the activation of the DEVDase in AKR-2B cells is prevented by numerous agents like PDGF, TPA, and DEVD.cmk (R. Schäfer, D. Karbach, and J. Hoppe, Exp. Cell Res. 240, 28--39). All these agents completely prevented PKC-epsilon cleavage, indicating a tight correlation between DEVDase activity and PKC-epsilon cleavage. By using recombinant caspase-3 or highly purified DEVDase from cytosolic extracts we localized by Edman degradation the cleavage site in recombinant PKC-epsilon to asp383 in the hinge region between regulatory and catalytic domains. The corresponding tetrapeptide sequences SSPD and SATD for human and mouse PKC-epsilon, respectively, are unusual for caspase-3. Expression of the catalytic domain or of the cleavage-resistant mutant D383A had no effect on cell death in AKR-2B cells.

Animals↗
PubMed2001

Novel modified adenosine 5'-triphosphate analogues pharmacologically characterized in human embryonic kidney 293 cells highly expressing rat brain P2Y(1) receptor: Biotinylated analogue potentially suitable for specific P2Y(1) receptor isolation.

Rat brain P2Y(1) (rP2Y(1)) receptor-transfected human embryonic kidney cells (HEK 293) were recently shown to have enhanced reactivity to both ATP and ADP (Vöhringer C, Schäfer R, Reiser G. Biochem Pharmacol 2000;59:791-800). Here, we demonstrated the usefulness of this cell line as a system for further studying novel adenine nucleotide analogues (Halbfinger et al. J Med Chem 1999;42:5325-37) and for the biochemical characterization of the P2Y(1) receptor. By measurement of intracellular Ca(2+) release, for 2-butylthio-, 2-butylamino-, and 2-butyloxy-ATP (2-BuS-, 2-BuNH-, 2-BuO-ATP), EC(50) values of 1.3, 5, and 60 nM were determined, markedly lower than the value for ATP (130 nM). The EC(50) for 2-BuSADP was 1.1 nM. The corresponding 8-substituted ATP analogues showed a substantially lower potency than ATP (ATP > 8-BuSATP > 8-BuNHATP approximately 8-BuOATP). AMP induced intracellular Ca(2+) release with a very low potency; 2- and 8-substitutions on AMP caused no significant potency shift, except for 2-BuSAMP (EC(50) = 180 nM). Another new P2Y receptor probe, 2-[(6-biotinylamido)-hexylthio]ATP, was 22-fold more potent than ATP (EC(50) = 6 nM), revealing that even more bulky substituents linked to the C-2 position bind with high affinity at the P2Y(1) receptor. This biotinylated probe was successfully used for the enrichment of the P2Y(1) receptor tagged with green fluorescent protein from a crude membrane fraction. This one-step enrichment provides a substantial advance for P2Y(1) receptor purification. Thus, human embryonic kidney 293 cells stably transfected with the rP2Y(1) receptor represent a powerful model system for pharmacological characterization of the P2Y(1) receptor, circumventing problems associated with natural systems. They provide a means for the development of P2Y(1) ligands of high potency and a good source for obtaining purified P2Y(1) receptor.

Adenosine Triphosphate↗
PubMed2001

The Kapandji technique for fixation of distal radius fractures--a biomechanical comparison of primary stability.

The goal of this study was to compare Kapandji-K-wiring and established K-wiring techniques of the distal radius fracture for primary stability in a biomechanical model: dorsal K-wiring according to Kapandji using different angles of the K-wire, parallel and diagonal alignment of the K-wires. A new testing system which uses a synthetic material enabled us to carry out the cantilever bending test. By application of a lower load, the Kapandji procedure shows a higher reactive torque and stiffness. A higher reaction force of the other techniques, especially of the parallel wiring, are only observable under high-grade bending stress. Application of the Kapandji procedure with K-wires at a smaller angle to the axis of the radius results in the highest primary stability of the procedures investigated in the essential range of initial deformation.

Biomechanical Phenomena↗
PubMed2001

Caveolin-1 is down-regulated in human ovarian carcinoma and acts as a candidate tumor suppressor gene.

To identify novel markers differentially expressed in ovarian cancer versus normal ovary, we hybridized microarrays with cDNAs derived from normal human ovaries and advanced stage ovarian carcinomas. This analysis revealed down-regulation of the caveolin-1 gene (CAV1) in ovarian carcinoma samples. Suppression of CAV1 in ovarian carcinomas was confirmed using a tumor tissue array consisting of 68 cDNA pools from different matched human tumor and normal tissues. Immunohistochemistry demonstrated expression of caveolin-1 in normal and benign ovarian epithelial cells, but loss of expression in serous ovarian carcinomas. In low-grade carcinomas, redistribution of caveolin-1 from a membrane-associated pattern observed in normal epithelium to a cytoplasmic localization pattern was observed. No expression of caveolin-1 was detectable in four of six ovarian carcinoma cell lines investigated. In SKOV-3 and ES-2 carcinoma cells, which express high levels of the caveolin-1 protein, phosphorylation of the 22-kd caveolin-1 isoform was detected. Inhibition of both DNA methylation and histone deacetylation using 5-aza-2'deoxycytidine and Trichostatin A, respectively, relieves down-regulation of caveolin-1 in OAW42 and OVCAR-3 cells which is in part mediated by direct regulation at the mRNA level. Expression of CAV1 in the ovarian carcinoma cell line OVCAR-3, resulted in suppression of tumor cell survival in vitro, suggesting that the CAV1 gene is likely to act as a tumor suppressor gene in human ovarian epithelium.

Carcinoma↗