Biomedical subjects

Gabriele Müller

Publications and source records attributed to Gabriele Müller.

10 recordsLinked to original sources

PubMed2007

Induced and repressed genes after irradiation sensitizing by pentoxyphylline.

Aim in cancer therapy is to increase the therapeutic ratio eliminating the disease while minimizing toxicity to normal tissues. Radiation therapy is a main component in targeting cancer. Radiosensitizing agents like pentoxyphylline (PTX) have been evaluated to improve radiotherapy. Commonly, cells respond to radiation by the activation of specific early and late response genes as well as by inhibition of genes, which are expressed under normal conditions. A display of the genetic distinctions at the level of transcription is given here to characterize the molecular events underlying the radiosensitizing mechanisms. The method of suppression subtractive hybridization allows the visualization of both induced and repressed genes in irradiated cells compared with cells sensitized immediately after irradiation. The genes were isolated by cDNA-cloning, differential analysis and sequence similarity search. Genes involved in protein synthesis, metabolism, proteolysis and transcriptional regulation were detected. It is important that genes like KIAA280, which were only known as unidentified EST sequences before without function, but inaccessible by array technology were recovered as functional genes. Database searches for PTX-induced genes detected a human mRNA completely unknown. In case of suppressed genes, we detected several mRNAs; one thereof shows homology to a hypothetical protein possibly involved in signal transduction. A further mRNA encodes the protein BM036 supposed to associate with the E2F transcription factor. A hypothetical protein H41 was detected, which may repress the Her-2/neu receptor influencing breast cancer, gliomas and prostate tumors. Radiation combined with PTX may lead to a better prognosis by down regulation of the Her-2/neu, which will be proven by clinical studies in the near future.

Cell Line, Tumor↗

PubMed2005

Two-hybrid fluorescence cross-correlation spectroscopy detects protein-protein interactions in vivo.

Fluorescence cross-correlation spectroscopy (FCCS) uses the correlated motion of two distinct fluorophores to detect their interaction. Whereas FCCS has been used with chemically or genetically labeled interaction partners in vitro, FCCS has never been demonstrated in vivo between two autofluorescent proteins. At least one reaction partner was always chemically labeled. Fos and Jun, two components of the AP-1 transcription factor, are known to exert their function as a dimer and can therefore serve as a reference for dimer formation. Expressing fusion proteins between Fos and the enhanced green fluorescent protein (EGFP), as well as Jun and the monomeric red fluorescent protein 1 (mRFP1) in HeLa cells, we show here, for the first time, in vivo FCCS detection of protein-protein interactions. The mobility of the dimerized species is slow, indicating that DNA-binding might stabilize dimerization. The technique has rich potential applications for the rapid screening of protein-protein interactions in vivo, which are able to clarify events during the whole life of cells.

Biophysics↗

PubMed2005

[Rigid RSA DMP for Type-2 Diabetes Mellitus: results of a three-state survey].

Diabetes contracts, such as those used in Saxony, were already excellent, effective diabetes programs in line with the concept of disease management. This was due to well-accepted practical guidelines and to an integrated care concept that was implemented with little bureaucratic effort through an internal quality-control management; this was based in turn on rapid, practicable documentation in the framework of interdisciplinary quality round tables. The present RSA DMP, in contrast, are to be fundamentally improved and simplified. This must occur also in view of the handling of guidelines, so that these do not jeopardize well-established and positively-evaluated healthcare programs (and along with that the quality of patient care) in the regions with well-functioning systems. In view of the persistent critiques on form and content of the state-regulated chronic-disease programs, the group "DMP in Central Germany", representing the German federal states of Saxony, SaxonyAnhalt, and Thuringia, initiated at the end of 2003 a structured survey for the evaluation of the RSA DMP Type 2 Diabetes among general practitioners and diabetes specialists. Particularly critical evaluations of the RSA DMP originate from the federal states with well-developed guideline-based diabetes care programs, for example in the new Eastern federal states, Nord Rhine-Westphalia, and Bavaria. With its minimal standards, the RSA DMP does not represent any progress in these regions and does not contribute to cost reduction. Detailed medical contents and professional guidelines do not belong to a law ordinance but to the already existing medical guidelines.

Delivery of Health Care↗

PubMed2004

Trichostatin A-induced histone acetylation causes decondensation of interphase chromatin.

The effect of trichostatin A (TSA)-induced histone acetylation on the interphase chromatin structure was visualized in vivo with a HeLa cell line stably expressing histone H2A, which was fused to enhanced yellow fluorescent protein. The globally increased histone acetylation caused a reversible decondensation of dense chromatin regions and led to a more homogeneous distribution. These structural changes were quantified by image correlation spectroscopy and by spatially resolved scaling analysis. The image analysis revealed that a chromatin reorganization on a length scale from 200 nm to >1 microm was induced consistent with the opening of condensed chromatin domains containing several Mb of DNA. The observed conformation changes could be assigned to the folding of chromatin during G1 phase by characterizing the effect of TSA on cell cycle progression and developing a protocol that allowed the identification of G1 phase cells on microscope coverslips. An analysis by flow cytometry showed that the addition of TSA led to a significant arrest of cells in S phase and induced apoptosis. The concentration dependence of both processes was studied.

Acetylation↗

PubMed2003

Counting nucleosomes in living cells with a combination of fluorescence correlation spectroscopy and confocal imaging.

Although methods for light microscopy of chromatin are well established, there are no quantitative data for nucleosome concentrations in vivo. To establish such a method we used a HeLa clone expressing the core histone H2B fused to the enhanced yellow fluorescent protein (H2B-EYFP). Quantitative gel electrophoresis and fluorescence correlation spectroscopy (FCS) of isolated oligonucleosomes show that 5% of the total H2Bs carry the fluorescent tag and an increased nucleosome repeat length of 204 bp for the fluorescent cells. In vivo, the mobility and distribution of H2B-EYFP were studied with a combination of FCS and confocal imaging. With FCS, concentration and brightness of nascent molecules were measured in the cytoplasm, while in the nucleoplasm a background of mobile fluorescent histones was determined by continuous photobleaching. Combining these results allows converting confocal fluorescence images of nuclei into calibrated nucleosome density maps. Absolute nucleosome concentrations in interphase amount up to 250 microM locally, with mean values of 140(+/-28)microM, suggesting that a condensation-controlled regulation of site accessibility takes place at length scales well below 200 nm.

Flow Cytometry↗

PubMed2003

Analyzing intracellular binding and diffusion with continuous fluorescence photobleaching.

Transport and binding of molecules to specific sites are necessary for the assembly and function of ordered supramolecular structures in cells. For analyzing these processes in vivo, we have developed a confocal fluorescence fluctuation microscope that allows both imaging of the spatial distribution of fluorescent molecules with confocal laser scanning microscopy and probing their mobility at specific positions in the cell with fluorescence correlation spectroscopy and continuous fluorescence photobleaching (CP). Because fluorescence correlation spectroscopy is restricted to rapidly diffusing particles and CP to slower processes, these two methods complement each other. For the analysis of binding-related contributions to mobility we have derived analytical expressions for the temporal behavior of CP curves from which the bound fraction and/or the dissociation rate or residence time at binding sites, respectively, can be obtained. In experiments, we investigated HeLa cells expressing different fluorescent proteins: Although enhanced green fluorescent protein (EGFP) shows high mobility, fusions of histone H2B with the yellow fluorescent protein are incorporated into chromatin, and these nuclei exhibit the presence of a stably bound and a freely diffusing species. Nonpermanent binding was found for mTTF-I, a transcription termination factor for RNA polymerase I, fused with EGFP. The cells show fluorescent nucleoli, and binding is transient. CP yields residence times for mTTF-I-EGFP of approximately 13 s.

Bacterial Proteins↗

PubMed2003

Water regulates oxygen binding in hagfish (Myxine glutinosa) hemoglobin.

Hagfish hemoglobin (Hb) is considered to represent a transition stage between invertebrate and vertebrate hemoglobins. The Hb system of Myxine glutinosa consists of three monomeric hemoglobins, which upon deoxygenation associate to form primarily heterodimers and heterotetramers. Myxine glutinosa is an osmoconformer, whose red blood cells show the exceptional ability to swell and remain swollen under hyposmotic conditions. In order to determine whether water activity regulates hemoglobin function, the effect of changes in osmolality on hemoglobin-O(2) affinity was investigated by applying the osmotic stress method to purified hemoglobins as well as intact red blood cells. Oxygen affinity decreases when water activity increases, indicating that water molecules stabilize the low-affinity, oligomeric state of the hemoglobin. This effect is opposite to that observed in tetrameric vertebrate hemoglobins, but resembles that seen in the dimeric hemoglobin of the marine clam Scapharca inaequivalvis. Our data show that water may act as an allosteric effector for hemoglobin within intact red cells and even in animals that do not experience large variations in blood osmolality.

Allosteric Regulation↗

PubMed2002

Clonal origin of multifocal renal cell carcinoma as determined by microsatellite analysis.

PURPOSE: The reported incidence of satellite tumor lesions in renal cell carcinoma (7% to 25%) suggests that there is a risk of local recurrence after nephron sparing surgery. It remains largely unknown whether small satellite tumors show malignant features and whether they are metastases from the primary tumor. Therefore, we determined the clonality of multifocal tumors by molecular genetic analysis. MATERIALS AND METHODS: A total of 19 multifocal clear cell renal cell carcinomas were investigated by microsatellite analysis using 6 markers for chromosome 3p, namely D3S1560, D3S1289, D3S1766, D3S1300, D3S1566 and D3S1663. Polymerase chain reaction was performed according to standard protocols, followed by gel electrophoresis and automated analysis using an automated DNA sequencer (Li-Cor, Lincoln, Nebraska). RESULTS: All primary clear cell tumors were characterized by loss of heterozygosity on 3p. Multifocal tumors showed identical microsatellite alterations with at least 1 marker in 17 of the 19 cases. In 2 cases different microsatellite patterns were detected in tumors from the same kidney. CONCLUSIONS: Identical loss of heterozygosity and shift patterns detected in different tumors in the same kidney strongly suggest that multifocal clear cell renal cell carcinomas have a common clonal origin in most cases. These findings indicate that satellite tumors are the result of intrarenal metastasis from the primary tumor. The clinical implications of these results must be correlated with the clinical disease course in patients with multifocal renal cell carcinoma.

Alleles↗

PubMed2002

Altered drug disposition of the platelet activating factor antagonist apafant in mdr1a knockout mice.

The aim of the present study was to determine a potential impact of P-glycoprotein (P-gp) on the tissue distribution and disposition of apafant (WEB 2086, CAS 105219-56-5), a selective platelet-activating factor antagonist, and on digoxin in mdr1a(-/-) and wildtype mice. Transport experiments in Caco-2 monolayers at low concentrations (<10 microM) showed that the secretory flux of [(14)C]apafant and [(3)H]digoxin exceeded the absorptive flux nine times. This efflux was concentration dependent and subject to inhibition by the P-gp substrates verapamil and cyclosporin A. This indicates that active drug transporter P-gp was involved in apafant and digoxin absorption. Mdr1a(-/-) mice showed a more than 70-fold higher concentration of digoxin-related radioactivity (P<0.001) in the brain than wildtype mice after intravenous doses of 0.05 mg/kg [(3)H]digoxin. Differences were less pronounced in other tissues. Both liquid scintillation counting and whole body autoradiography yielded comparable results and they also matched recently published data. Apafant-related radioactivity was about ten-fold higher in the brain of mdr1a(-/-) mice compared to wildtype mice following intravenous doses of 2 mg/kg radiolabelled apafant. Only slight or negligible differences were observed in other tissues. In wildtype mice, intestinal excretion of [(14)C]apafant (54.9%) exceeded biliary excretion (26.5%). However, in mdr1a(-/-) mice biliary excretion (50.7%) exceeded intestinal excretion (6.8%). These differences were mirrored in the urinary and faecal excretion. Pharmacokinetic parameters of apafant and radioactivity did not differ between wildtype and mdr1a(-/-) mice. The conclusions were: (1) apafant and digoxin are P-gp substrates, and (2) absence of mdr1a encoded P-gp significantly alters tissue distribution (especially in brain) and excretion routes (biliary and intestinal) of apafant.

ATP Binding Cassette Transporter, Subfamily B↗

PubMed2002

Interleukin-10-treated dendritic cells modulate immune responses of naive and sensitized T cells in vivo.

Interleukin-10 is a pleiotropic cytokine known to have inhibitory effects on the accessory functions of dendritic cells. In vitro, interleukin-10 converts immature dendritic cells into tolerizing antigen- presenting cells. To assess whether interleukin-10-treated dendritic cells exert tolerizing effects in vivo, CD4+ T cells from DO11.10 ovalbumin-T cell receptor transgenic mice were transferred to syngeneic BALB/c recipients. Recipient animals were treated with ovalbumin-pulsed/unpulsed, interleukin-10-treated/untreated CD11c+ dendritic cells thereafter and ovalbumin-specific proliferation of lymph node cells was assessed by restimulation with the peptide in vitro. In prophylactic experiments, recipients received naive CD4+ DO11.10 T cells and were immunized with ovalbumin323-339 peptide in incomplete Freund's adjuvant after treatment with various subtypes of dendritic cells. Strong ovalbumin-specific proliferation was observed in animals immunized with control ovalbumin-dendritic cells. Minimal proliferation was found in mice treated with ovalbumin-pulsed, interleukin-10-treated dendritic cells. In therapeutic experiments, preactivated CD4+ DO11.10 T cells were transferred, and recipients were treated with dendritic cells as described. Ovalbumin-specific proliferation was strong in recipients treated with ovalbumin-dendritic cells. CD4+ T cell proliferation from ovalbumin-interleukin-10-dendritic cell treated animals was below background. When delayed type hypersensitivity reactions in the footpads of prophylactically or therapeutically vaccinated animals were tested, mice treated with ovalbumin-interleukin-10-dendritic cells showed no footpad swelling compared with controls. Rechallenge with the antigen in vitro and in vivo did not alter the inhibitory effect of interleukin-10-treated dendritic cells. Thus, interleukin-10-treated dendritic cells inhibit ovalbumin-specific immune responses in naive and sensitized mice.

Animals↗