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R Schäfer

Publications and source records attributed to R Schäfer.

At least 19 recordsLinked to original sources
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↗
PubMed2001

[Factoral structure and psychometric properties of the German version of the Toronto Alexithymia Scale (TAS-20) in psychosomatic patients].

The present paper describes the attempt to cross-validate the three-factorial structure of the German Version of the 20-Item Toronto Alexithymia Scale in a population of 419 patients, suffering of psychogenic disorders. However, results neither revealed good internal consistencies nor was it possible to obtain an acceptable adaptation of the empirical data on the postulated model. Therefore an explorative factor analysis was performed in order to get a more consistent factor model. The hereby resulting four-factorial structure was congruent with reference to theoretical and empirical data of Alexithymia. However, additional cross-validation in other populations has to be investigated.

Adult↗
PubMed2001

ras oncogene expression determines sensitivity for intercellular induction of apoptosis.

Fibroblasts carrying an inducible ras oncogene acquire the transformed phenotype after oncogene induction. As a consequence, the transformed cells become sensitive to intercellular induction of apoptosis, a novel regulatory process directed by non-transformed fibroblasts against their transformed descendants. The causal relationship between oncogene expression and sensitivity to intercellular induction of apoptosis is based on extracellular superoxide anion production by oncogene-expressing cells. Superoxide anions (after dismutation to hydrogen peroxide) thereby foster HOCl synthesis and at the same time direct the selectivity of apoptosis induction through hydroxyl generation from HOCl. In parallel, ras expression enhances the sensitivity of fibroblasts for apoptosis-inducing stimuli like cycloheximide, ceramide and mitomycin C. This sensitization seems to be based on a decreased concentration of short lived endogenous apoptosis inhibitors. TGF-beta, like ras induction, decreases the concentration of endogenous apoptosis inhibitors, but does not induce the transformed phenotype. Therefore, TGF-beta treatment alone is not sufficient to render fibroblasts sensitive for intercellular induction of apoptosis, but TGF-beta treatment in parallel with ras activation enhances intercellular induction of apoptosis. Our findings demonstrate that Ras-mediated superoxide anion production determines sensitivity to intercellular induction of apoptosis, whereas the parallel decrease in endogenous apoptosis inhibitors modulates the kinetics of apoptosis induction.

Animals↗
PubMed2001

Sonic hedgehog is a survival factor for hypaxial muscles during mouse development.

Sonic hedgehog (Shh) has been proposed to function as an inductive and trophic signal that controls development of epaxial musculature in vertebrate embryos. In contrast, development of hypaxial muscles was assumed to occur independently of Shh. We here show that formation of limb muscles was severely affected in two different mouse strains with inactivating mutations of the Shh gene. The limb muscle defect became apparent relatively late and initial stages of hypaxial muscle development were unaffected or only slightly delayed. Micromass cultures and cultures of tissue fragments derived from limbs under different conditions with or without the overlaying ectoderm indicated that Shh is required for the maintenance of the expression of myogenic regulatory factors (MRFs) and, consecutively, for the formation of differentiated limb muscle myotubes. We propose that Shh acts as a survival and proliferation factor for myogenic precursor cells during hypaxial muscle development. Detection of a reduced but significant level of Myf5 expression in the epaxial compartment of somites of Shh homozygous mutant embryos at E9.5 indicated that Shh might be dispensable for the initiation of myogenesis both in hypaxial and epaxial muscles. Our data suggest that Shh acts similarly in both somitic compartments as a survival and proliferation factor and not as a primary inducer of myogenesis.

Adaptor Proteins, Signal Transducing↗
PubMed2000

Selective downregulation of VEGF-A(165), VEGF-R(1), and decreased capillary density in patients with dilative but not ischemic cardiomyopathy.

Cardiomyopathy (CM) comprises a heterogeneous group of diseases, including ischemic (ICM) and dilative (DCM) forms. The pathogenesis of primary DCM is not clearly understood. Recent studies in mice show that vascular endothelial growth factor (VEGF) is involved in ICM. Whether VEGF plays a role in human CM is unknown. We examined the mRNA and protein expression of VEGF and its receptors in hearts of patients with end-stage DCM and ICM and in healthy individuals using real-time polymerase chain reaction and Western blotting. Number of capillaries, area of myocytes, and collagen were calculated in cardiac biopsies using transmission electron microscopy. In DCM, except for VEGF-C, mRNA transcript levels of VEGF-A(165), VEGF-A(189), and VEGF-B and the protein level of VEGF-A and VEGF-R(1) were downregulated compared with controls (P:<0.05). However, in ICM, mRNA transcript levels of VEGF isoforms and protein levels of VEGF-C were upregulated. The vascular density was decreased in DCM but increased in ICM compared with controls (P:<0. 05). Muscular hypertrophy was not different for ICM and DCM, although DCM had more collagen (P:<0.05). Blunted VEGF-A and VEGF-R(1) protein expression and downregulated mRNA of the predominant isoform of VEGF-A, VEGF-A(165), to our knowledge shown here for the first time, provide evidence that the VEGF-A defect in DCM is located upstream. Whether downregulation of certain VEGF isoforms in DCM is a cause or consequence of this disorder remains unclear, although upregulated VEGF levels in ICM are most likely the result of ischemia.

Adult↗
PubMed2000

[Asymptomatic diclofenac-induced acute hepatitis].

HISTORY AND ADMISSION FINDINGS: After removal of an herniated intervertebral disc a 49-year-old man had recurrent pain in the lumbar region, treated with diclofenac and flupirtine. 4 months postoperatively he was referred to the author's hospital because of progressive rise in transaminase levels and in cholestasis parameters. Physical examination on admissions was unremarkable, except for jaundice. INVESTIGATIONS: Laboratory tests showed an increases in transaminases (GPT 769 U/l [normal: < 23 U/l], GOT 285 U/l [normal: < 19 U/l]) and in cholestasis-related values (gamma-GT 172 U/l [Norm 6-28 U/l], alkaline phosphatase 207 U/l [normal < 175 U/l], total bilirubin: 2.5 mg/dl [Norm < 1.1 mg/dl]). This corresponded to the histological finding of toxic damage to the liver parenchyma. Viral, autoimmune and metabolic causes had been excluded. DIAGNOSES, TREATMENT AND COURSE: The history as well as laboratory and histological findings indicated diclofenac-induced toxic hepatitis. The drug was discontinued and without any treatment all laboratory values returned to normal within 9 weeks. CONCLUSION: Diclofenac, a frequently prescribed non-steroid anti-inflammatory drug, is generally thought to be relatively safe. But it can produce toxic hepatitis which may be asymptomatic (but this patient was jaundiced) and may take an unforeseeable course. This should be taken account of by regularly monitoring liver functions.

Acute Disease↗
PubMed2000

Gene expression profiling of advanced lung cancer.

Lung cancer is a highly aggressive neoplasm with 85% mortality. To identify new tumor-associated genes, we compared the expression profile of a primary metastasizing adenocarcinoma with normal airway epithelial cells. Two cDNA libraries of up- and downregulated genes were generated, comprising 253 and 299 clones, respectively. The sequence analysis revealed 205 different known genes and 314 cDNA fragments of unknown functions. Northern-blot analysis of 167 clones confirmed differential expression in 58%, and indicated a similar expression pattern in additional lung-cancer cell lines for selected clones, strengthening the value of this model for the identification of new candidate genes in lung carcinogenesis.

Chromosome Mapping↗
PubMed2000

A chimeric rat brain P2Y1 receptor tagged with green-fluorescent protein: high-affinity ligand recognition of adenosine diphosphates and triphosphates and selectivity identical to that of the wild-type receptor.

We tested how the green fluorescent protein (GFP) tag affects signaling of the nucleotide-activated P2Y1 receptor. Therefore, we generated stably transfected human embryonic kidney 293 cells expressing the rat P2Y1 wild-type receptor (rP2Y1-wt) or the receptor tagged at the C-terminus with the enhanced GFP (rP2Y1-eGFP). The chimeric rP2Y1-eGFP receptor is localized mainly to the plasma membrane as revealed by Western blotting of subcellular fractions. Both receptors were analyzed by measuring Ca2+ responses to short pulses of the agonists in single cells by continuous superfusion with medium. The rP2Y1-eGFP receptor was coupled to Ca2+ release as was the rP2Y1-wt receptor. 2-Methylthio adenosine 5'-diphosphate and -triphosphate (2-MeSATP and 2-MeSADP) were the most potent agonists at the heterologously expressed receptors, with EC50 values of 50 to 70 nM for rP2Y1-eGFP and 0.06 to 0.4 nM for rP2Y1-wt. These potencies of the two P2Y-selective agonists at rP2Y1-wt receptor-expressing cells are the highest values reported so far. This increase is probably due to a receptor reserve. In both rP2Y1-wt- and in rP2Y1-eGFP-expressing cells, the effect of 2-MeSATP was inhibited equally by the antagonist pyridoxal phosphate-6-azophenyl-2',4'-disulfonic acid. We established that ATP as well as adenosine 5'-O-(1-thiotriphosphate) (ATPalphaS) are full agonists at the rP2Y1 receptor at both transfected cell lines. The rP2Y1-eGFP receptor has the same ligand selectivity as the rP2Y1-wt receptor (2-MeSADP = 2-MeSATP > ADP > ATPalphaS, ATP >> UTP). Thus, the GFP-tagged P2Y1 receptor is fully active and shows regular signal transduction coupling. It provides the means for biochemical characterization, since it can be solubilized and is a tool for further physiological analysis.

Adenosine Diphosphate↗
PubMed2000

Purification and characterization of a magnesium-dependent neutral sphingomyelinase from bovine brain.

The magnesium-dependent, plasma membrane-associated neutral sphingomyelinase (N-SMase) catalyzes hydrolysis of membrane sphingomyelin to form ceramide, a lipid signaling molecule implied in intracellular signaling. We report here the biochemical purification to apparent homogeneity of N-SMase from bovine brain. Proteins from Nonidet P-40 extracts of brain membranes were subjected to four purification steps yielding a N-SMase preparation that exhibited a specific enzymatic activity 23,330-fold increased over the brain homogenate. When analyzed by two-dimensional gel electrophoresis, the purified enzyme presented as two major protein species of 46 and 97 kDa, respectively. Matrix-assisted laser desorption/ionization-mass spectrometry analysis of tryptic peptides revealed at least partial identity of these two proteins. Amino acid sequencing of tryptic peptides showed no apparent homologies of bovine N-SMase to any known protein. Peptide-specific antibodies recognized a single 97-kDa protein in Western blot analysis of cell lysates. The purified enzyme displayed a K(m) of 40 microM for sphingomyelin with an optimal activity at pH 7-8. Bovine brain N-SMase was strictly dependent on Mg(2+), whereas Zn(2+) and Ca(2+) proved inhibitory. The highly purified bovine N-SMase was effectively blocked by glutathione and scyphostatin. Scyphostatin proved to be a potent inhibitor of N-SMase with 95% inhibition observed at 20 microM scyphostatin. The results of this study define a N-SMase that fulfills the biochemical and functional criteria characteristic of the tumor necrosis factor-responsive membrane-bound N-SMase.

Amides↗