Biomedical subjects

Christian Scholz

Publications and source records attributed to Christian Scholz.

At least 19 recordsLinked to original sources

PubMed2006

Re-programming of newt cardiomyocytes is induced by tissue regeneration.

Newt hearts are able to repair substantial cardiac injuries without functional impairment, whereas mammalian hearts cannot regenerate. The cellular and molecular mechanisms that control the regenerative capacity of the newt heart are unknown. Here, we show that the ability of newt cardiomyocytes to regenerate cardiac injuries correlates with their ability to transdifferentiate into different cell types. Mechanical injury of the heart led to a severe reduction of sarcomeric proteins in the myocardium, indicating a partial de-differentiation of adult newt cardiomyocytes during regeneration. Newt cardiomyocytes implanted into regenerating limbs lost their cardiac phenotype and acquired skeletal muscle or chondrocyte fates. Reprogramming of cardiomyocytes depended on contact with the limb blastema because cardiomyocytes implanted into intact, non-regenerating limbs or cultured in vitro retained their original identity. We reason that signals from the limb blastema led to de-differentiation of cardiomyocytes, cell proliferation and re-differentiation into specialized cells and propose that the ability of cardiomyocytes to transdifferentiate into different cell types reflects the cellular program that enables heart regeneration.

Animals↗

PubMed2006

One step cloning of defined DNA fragments from large genomic clones.

Recently, the nucleotide sequences of entire genomes became available. This information combined with older sequencing data discloses the exact chromosomal location of millions of nucleotide markers stored in the databases at NCBI, EMBO or DDBJ. Despite having resolved the intron/exon structures of all described genes within these genomes with a stroke of a pen, the sequencing data opens up other interesting possibilities. For example, the genomic mapping of the end sequences of the human, murine and rat BAC libraries generated at The Institute for Genomic Research (TIGR), reveals now the entire encompassed sequence of the inserts for more than a million of these clones. Since these clones are individually stored, they are now an invaluable source for experiments which depend on genomic DNA. Isolation of smaller fragments from such clones with standard methods is a time consuming process. We describe here a reliable one-step cloning technique to obtain a DNA fragment with a defined size and sequence from larger genomic clones in less than 48 hours using a standard vector with a multiple cloning site, and common restriction enzymes and equipment. The only prerequisites are the sequences of ends of the insert and of the underlying genome.

Animals↗

PubMed2006

Selective upregulation of beta1-adrenergic receptors and dephosphorylation of troponin I in end-stage heart failure patients supported by ventricular assist devices.

In terminal failing hearts, adrenergic receptors are downregulated and intracellular adrenergic signal transduction is inhibited. Mechanical circulatory support by ventricular assist devices (VAD) is used to bridge patients to heart transplantation. Mechanical unloading by VAD may induce reverse remodeling in heart transplantation (HTx) candidates. However, little is known on beta-adrenergic receptor subtype regulation and adrenergic signal transduction under VAD-support. We investigated paired myocardial samples from 16 VAD-supported patients and 9 non-failing donor hearts. We analyzed beta-adrenergic receptor subtype regulation by real-time PCR and radioligand binding and cardiac troponin I phosphorylation (by phospho-cTnI-specific antibodies). We found that the beta1-adrenergic receptor (beta1AR) is downregulated at VAD-implantation on mRNA and protein levels whereas the beta2-adrenergic receptor (beta2AR) was not. After VAD-support, beta1AR protein but not its mRNA was upregulated, whereas the degree of cTnI-phosphorylation was reduced. Upregulation of beta1AR was enhanced by beta blocking medication during VAD-support. However, in 9 out of 15 patients, beta1AR-density remained below the 0.25 percentile of donor hearts. VAD-support is associated with partial normalization of the betaAR-signal transduction pathways. This beneficial effect is related to a posttranscriptional increase in beta1AR-density.

Adult↗

PubMed2006

SlyD proteins from different species exhibit high prolyl isomerase and chaperone activities.

SlyD is a putative folding helper protein from the Escherichia coli cytosol, which consists of an N-terminal prolyl isomerase domain of the FKBP type and a presumably unstructured C-terminal tail. We produced truncated versions without this tail (SlyD) for SlyD from E. coli, as well as for the SlyD orthologues from Yersinia pestis, Treponema pallidum, Pasteurella multocida, and Vibrio cholerae. They are monomeric in solution and unfold reversibly. All SlyD variants catalyze the proline-limited refolding of ribonuclease T1 with very high efficiencies, and the specificity constants (kcat/KM) are equal to approximately 10(6) M(-1) s(-1). These large values originate from the high affinities of the SlyD orthologues for unfolded RCM-T1, which are reflected in low KM values of approximately 1 microM. SlyD also exhibits pronounced chaperone properties. Permanently unfolded proteins bind with high affinity to SlyD and thus inhibit its prolyl isomerase activity. The unfolded protein chains do not need to contain proline residues to be recognized and bound by SlyD. The conservation of prolyl isomerase activity and chaperone properties within the SlyD family suggests that these proteins might act as true folding helpers in the bacterial cytosol. The SlyD proteins are also well suited for biotechnological applications. As fusion partners they facilitate the refolding and increase the solubility of aggregation-prone proteins such as the gp41 ectodomain fragment of HIV-1.

Amino Acid Sequence↗

PubMed2005

Arsenic trioxide induces regulated, death receptor-independent cell death through a Bcl-2-controlled pathway.

Arsenic trioxide (As2O3, arsenite) efficiently kills cells from various hematologic malignancies and has successfully been employed especially for the treatment of acute promyelocytic leukemia. There and in lymphoid cells, we demonstrated that As2O3 induces cell death in a caspase-2- and -9-independent fashion. Here, we address a potential role of death receptor signaling through the FADD/caspase-8 death-inducing signaling complex in As2O3-induced cell death. In detail, we demonstrate that As2O3 induces cell death independently of caspase-8 or FADD and cannot be blocked by disruption of CD95/Fas receptor ligand interaction. Unlike in death receptor ligation-induced apoptosis, As2O3-induced cell death was not blocked by the broad-spectrum caspase inhibitor z-VAD-fmk or the caspase-8-specific inhibitor z-IETD-fmk. Nevertheless, As2O3-induced cell death occurred in a regulated manner and was abrogated upon Bcl-2 overexpression. In contrast, As2O3-induced cell demise was neither blocked by the caspase-9 inhibitor z-LEHD-fmk nor substantially inhibited through the expression of a dominant negative caspase-9 mutant. Altogether our data demonstrate that As2O3-induced cell death occurs independently of the extrinsic death receptor pathway of apoptosis. Cell death proceeds entirely via an intrinsic, Bcl-2-controlled mitochondrial pathway that does, however, not rely on caspase-9.

Adaptor Proteins, Signal Transducing↗

PubMed2005

Necrotic death but not irradiation abolishes costimulation of T-cell effector functions and survival by CD80-expressing tumor cells.

Tumor vaccination by the use of gene-modified cancer cells that provide costimulatory signals has been successfully applied in preclinical animal models and is currently evaluated in a variety of clinical settings. In previous work, we demonstrated the efficacy of B7.1/CD80 to promote tumor immunity in syngeneic murine models and to prevent deletion of activated T cells by activation-induced cell death (AICD). In clinical trials, tumor cell vaccines are generally inactivated to avoid transfer of live tumor cells, i.e., additional tumor burden. Previous data indicated, however, that inactivation of tumor cells by lethal ionizing irradiation abrogates tumor vaccination by CD80-expressing cells. Here, we compare living and irradiated allogeneic tumor cells regarding their capacity to induce T-cell effector functions and their propensity to interfere with T-cell deletion by apoptosis. Both lethally irradiated and nonirradiated tumor cells facilitated T-cell proliferation, tumor cell lysis, and interfered with T-cell AICD to a similar extent. In contrast, necrotic tumor cells failed to costimulate T-cell effector functions. Thus, irradiation does not seem to hamper tumor cell-mediated costimulation of T-cell effector functions. In contrast, necrosis of gene-modified tumor cells abrogates costimulation of T cells by CD80-expressing cells.

B7-1 Antigen↗

PubMed2005

Arsenic trioxide triggers a regulated form of caspase-independent necrotic cell death via the mitochondrial death pathway.

Cell death is generally believed to occur either by accidental, lytic necrosis or by programmed cell death, that is, apoptosis. The initiation and execution of cell death, however, is far more complex and includes pathways like caspase-independent apoptosis or actively triggered necrosis. In this study, we investigated the mechanisms of cell death induced by arsenic trioxide (arsenite, As2O3), a clinically efficient agent in anticancer therapy. As2O3-induced cell death coincides with cytochrome c release, facilitates mitochondrial permeability transition and is sensitive to inhibition by Bcl-x(L), indicating that cell demise is regulated through the mitochondrial apoptosis pathway. Nevertheless, only little caspase-3 activation was observed and As2O3-induced cell death was only weakly obstructed by the broad spectrum caspase inhibitor z-VAD-fmk. Moreover, disruption of caspase-9 or -2 failed to decrease the amount of As2O3-mediated cell death. Interestingly, As2O3-induced cell death had a predominantly necrosis-like phenotype as assessed by Annexin-V/propidium iodide staining and LDH release. Finally, blocking glutathione synthetase by buthionine sulfoximine enhanced the As2O3-mediated necrosis-like cell death without increasing caspase-3 cleavage. As2O3 does, however, not directly inhibit caspases, but appears to interfere with caspase activation. Altogether, our data clearly delineate a mode of As2O3-triggered cell death that differs considerably from that induced by conventional anticancer drugs. These findings may explain the capability of As2O3 to efficiently kill even chemoresistant tumor cells with disturbed apoptosis signaling and caspase activation, a frequent finding in malignancy.

Apoptosis↗

PubMed2005

Docetaxel and continuous-infusion fluorouracil versus epirubicin, cisplatin, and fluorouracil for advanced gastric adenocarcinoma: a randomized phase II study.

PURPOSE: A combination of docetaxel and fluorouracil (DF) was evaluated in an outpatient setting and compared with epirubicin, cisplatin, and fluorouracil (ECF), which served as an internal control arm to avoid selection bias. PATIENTS AND METHODS: Patients with metastatic or locally advanced gastric adenocarcinoma without prior chemotherapy were randomly assigned to receive either ECF (epirubicin 50 mg/m(2) day 1, cisplatin 60 mg/m(2) day 1, and fluorouracil 200 mg/m(2) days 1 through 21, every 3 weeks) or DF (docetaxel 75 mg/m(2) day 1, and fluorouracil 200 mg/m(2) days 1 through 21, every 3 weeks). RESULTS: Ninety patients were randomly assigned. Toxicity was rarely severe. Major toxic effects included diarrhea, stomatitis, and leukopenia in the DF arm and nausea, vomiting, and leukopenia in the ECF arm. Forty-three of 45 patients in each arm were assessable. In the DF arm, two patients (4.4%, intent to treat) experienced a confirmed complete tumor remission as best response, and 15 patients (33.3%) experienced a confirmed partial remission (overall response rate [ORR], 37.8%; 95% CI, 25.9% to 51.9%). Two patients (4.4%) in the ECF arm showed confirmed complete remission, and 14 (31.1%) showed confirmed partial remission (ORR, 35.6%; 95% CI, 24.8% to 48.7%). For the DF and ECF arms, the median survival was 9.5 and 9.7 months, and the median time to tumor progression 5.5 and 5.3 months, respectively. CONCLUSION: DF can be safely given in an ambulant setting. Compared with ECF, the dual combination of DF shows promising efficacy and may be an alternative treatment option that avoids cisplatin.

Adenocarcinoma↗

PubMed2005

Costimulation by CD137/4-1BB inhibits T cell apoptosis and induces Bcl-xL and c-FLIP(short) via phosphatidylinositol 3-kinase and AKT/protein kinase B.

Costimulation is essential for induction of T lymphocyte proliferation and inhibition of activation-induced cell death. While signaling pathways activated following the ligation of the costimulatory molecule CD28 are well defined, less is known about the molecular events induced by alternative costimulators. CD137/4-1BB, a costimulatory member of the tumor necrosis factor receptor family, plays an important role during late primary T cell stimulation. Here, we demonstrate for the first time that inhibition of activation-induced cell death by exposure to the CD137/4-1BB ligand involves up-regulation of the anti-apoptotic protein c-FLIP(short). Inhibition of T cell death by 4-1BB ligation and up-regulation of c-FLIP(short) and Bcl-x(L) were abolished by blocking the phosphatidylinositol 3-kinase or the AKT/protein kinase B, which also mediate CD28-induced inhibition of activation-induced cell death. Our findings, therefore, demonstrate that costimulatory molecules, although belonging to different protein families and participating in distinct upstream signaling pathways, employ common downstream signaling pathways.

Animals↗

PubMed2005

Engrailed genes control developmental fate of serotonergic and noradrenergic neurons in mid- and hindbrain in a gene dose-dependent manner.

In vertebrates and insects, the homeobox transcription factors of the engrailed family have a dual function. They take part in regionalization during early embryogenesis and later in neuronal specification. In mammals, two engrailed homologues exist, engrailed-1 and engrailed-2, which are expressed in a broad band around the isthmus at an age when the serotonergic and noradrenergic neurons in mid/hindbrain are generated. The analysis of engrailed-1 and -2 double mutant mice revealed a specific, redundant, and gene dose-dependent requirement of the two transcription factors for the development of the serotonergic dorsal raphe nucleus and the noradrenergic locus caeruleus. Both nuclei are lost in engrailed double mutant mice; however, directly adjacent nuclei of the same neurotransmitter phenotype are not affected. An almost identical phenotype is found in mutant mice null for Wnt1, indicating that the engrailed genes provide essential positional information for the development of the two nuclei during early embryogenesis.

Animals↗

PubMed2004

Functional solubilization of aggregation-prone HIV envelope proteins by covalent fusion with chaperone modules.

The envelope proteins of human immunodeficiency virus (HIV) and human T-cell lymphotrophic virus (HTLV) mediate cell attachment and membrane fusion. For HIV-1, the precursor protein gp160 is cleaved proteolytically into two fragments, the surface-associated receptor binding subunit gp120 and the membrane spanning subunit gp41, which is involved in membrane fusion during virus entry. Soluble and immunoreactive variants of gp41 are essential for the reliable diagnosis of HIV-1 infections. Hitherto, gp41 was solubilized by adding detergents, or in acidic or alkaline solvents. We find that covalent fusions with SlyD or FkpA, two homodimeric Escherichia coli chaperones with peptidyl-prolyl isomerase activity, solubilize retroviral envelope proteins without compromising their immunological reactivity. gp41 from HIV-1, gp36 from HIV-2 and gp21 from HTLV could be expressed in large amounts in the Escherichia coli cytosol when fused with one or two subunits of SlyD or FkpA. The fusion proteins could be easily isolated and refolded, and showed high solubility and immunoreactivity, thus providing sensitive and reliable tools for diagnostic applications. Covalent fusions with SlyD or FkpA might be valuable generic tools for the solubilization and activation of aggregation-prone proteins.

Amino Acid Sequence↗

PubMed2004

Distinction of acute lymphoblastic leukemia from acute myeloid leukemia through microarray-based DNA methylation analysis.

Patterns of DNA methylation are substantially altered in malignancies compared to normal tissue, with both genome-wide hypomethylation and regional increase of cytosine methylation at dinucleotides of cytosine and guanine, i.e., CpG dinucleotides. While genome-wide hypomethylation renders chromosomes instable, hypermethylation of CpGs in promoter regions is generally associated with transcriptional silencing, e.g., of tumor suppressor genes. To investigate whether disease-specific methylation profiles exist for different entities of acute leukemia, a microarray-based DNA methylation analysis simultaneously assessing 249 CpG dinucleotides originating from 57 genes was employed. Hereby, samples from precursor B-cell acute lymphoblastic leukemia (ALL) could be distinguished from cases of acute myeloid leukemia by virtue of N33, EGR4, CDC2, CCND2, or MOS hypermethylation in ALL.

Acute Disease↗

PubMed2004

Suggested protocol for collecting, handling and preparing peat cores and peat samples for physical, chemical, mineralogical and isotopic analyses.

For detailed reconstructions of atmospheric metal deposition using peat cores from bogs, a comprehensive protocol for working with peat cores is proposed. The first step is to locate and determine suitable sampling sites in accordance with the principal goal of the study, the period of time of interest and the precision required. Using the state of the art procedures and field equipment, peat cores are collected in such a way as to provide high quality records for paleoenvironmental study. Pertinent field observations gathered during the fieldwork are recorded in a field report. Cores are kept frozen at -18 degree C until they can be prepared in the laboratory. Frozen peat cores are precisely cut into 1 cm slices using a stainless steel band saw with stainless steel blades. The outside edges of each slice are removed using a titanium knife to avoid any possible contamination which might have occurred during the sampling and handling stage. Each slice is split, with one-half kept frozen for future studies (archived), and the other half further subdivided for physical, chemical, and mineralogical analyses. Physical parameters such as ash and water contents, the bulk density and the degree of decomposition of the peat are determined using established methods. A subsample is dried overnight at 105 degree C in a drying oven and milled in a centrifugal mill with titanium sieve. Prior to any expensive and time consuming chemical procedures and analyses, the resulting powdered samples, after manual homogenisation, are measured for more than twenty-two major and trace elements using non-destructive X-Ray fluorescence (XRF) methods. This approach provides lots of valuable geochemical data which documents the natural geochemical processes which occur in the peat profiles and their possible effect on the trace metal profiles. The development, evaluation and use of peat cores from bogs as archives of high-resolution records of atmospheric deposition of mineral dust and trace elements have led to the development of many analytical procedures which now permit the measurement of a wide range of elements in peat samples such as lead and lead isotope ratios, mercury, arsenic, antimony, silver, molybdenum, thorium, uranium, rare earth elements. Radiometric methods (the carbon bomb pulse of (14)C, (210)Pb and conventional (14)C dating) are combined to allow reliable age-depth models to be reconstructed for each peat profile.

Environmental Monitoring↗

PubMed2003

Interaction of trigger factor with the ribosome.

The trigger factor of Escherichia coli is a prolyl isomerase and a chaperone. It interacts with the ribosome and affects the folding of newly formed protein chains. Therefore, the dynamics of the interactions of trigger factor with the ribosome and with unfolded protein chains should be tailored for this function. Previously, we had found that binding of unfolded proteins to trigger factor is fast and that the lifetime of the complex between these two components is only about 100 ms. Here, we have labeled the trigger factor in its amino-terminal, ribosome-binding domain with a fluorescent dye and investigated how it interacts with the ribosome. We found that this association, as well as the dissociation of the complex, are rather slow processes. The average lifetime of the complex is about 30 seconds (at 20 degrees C). The strong differences in the dynamics of the interactions of trigger factor with the ribosome and with protein substrates might ensure that, on the one hand, trigger factor remains bound to the ribosome while a protein chain is being synthesized, and, on the other hand, allows it to scan the newly formed protein for prolyl bonds that need catalysis of isomerization.

2-Naphthylamine↗

PubMed2003

Development and clinical application of a fast superposition algorithm in radiation therapy.

BACKGROUND AND PURPOSE: Dose calculation algorithms play a central role for the optimization and verification of treatment plans in radiation therapy. Complex treatment techniques like intensity modulated radiotherapy (IMRT) require accurate and fast dose algorithms especially for clinical cases which involve severe tissue inhomogeneities. For these cases the standard dose engine in current treatment planning systems--the convolution of photon pencil beams--usually fails to predict the dose with the required accuracy. The role of more accurate but time consuming dose calculations like superposition algorithms or Monte Carlo simulations in clinical practice is under investigation at several therapy centers. PATIENTS AND METHODS: The paper presents the design, implementation and the first application of a superposition algorithm in a clinical setting at the German Cancer Research Center (DKFZ). It first describes in detail how the superposition algorithm is adapted to the dose delivery system at DKFZ in terms of standard dosimetric data. Then details of the implementation of the algorithm are given with a focus on various methods for the reduction of dose computation times. Next, the algorithm is evaluated in various experiments with dosimetric phantoms. These studies are employed for the development of time efficient sampling strategies of the elemental dose kernels. Finally, the algorithm is applied to dose calculations of clinical cases with tumors adjacent to lung tissue. RESULTS: Severe differences in dose coverage of the tumors and dose burden of the surrounding tissues in comparison to standard pencil beam calculations are observed. A standard 4-7 beam plan in a convenient dose grid (approximately 3 mm in each direction) is calculated in about 30 min on a Pentium 4 (1.9 GHz) applying the superposition algorithm described here.

Algorithms↗

PubMed2003

Comparison of IMRT optimization based on a pencil beam and a superposition algorithm.

To investigate the role of sophisticated dose calculation methods for treatment planning, we compared conventional pencil beam optimized 6 and 15 MV intensity-modulated treatment plans with optimizations based on the superposition technique. Five lung and five head and neck IMRT cases with spatial resolutions of bixels and dose voxels usually employed in clinical practice were considered for tumor volumes between 15 and 500 cm3. We investigated the systematic error of the pencil beam algorithm and the pencil beam induced error to the optimal solution of bixel weights. For the lung cases, the pencil beam overestimated the mean dose deposited inside the planning target volume (PTV) by about 8%, for small lung tumors even up to 20.6%. In the head and neck cases only a slight overestimation in mean PTV dose of 1.5% was observed. The optimization with the superposition method substantially improved the dose coverage of the considered radiation targets. Additionally, for the head and neck cases, the brainstem was significantly spared by about 4% mean PTV dose through the use of the superposition technique. Our studies showed that, in target regions with intricate tissue inhomogeneities, superposition or Monte Carlo techniques have to be used for the optimization and the final dose calculation of intensity-modulated treatment plans.

Algorithms↗

PubMed2002

Tumour class prediction and discovery by microarray-based DNA methylation analysis.

Aberrant DNA methylation of CpG sites is among the earliest and most frequent alterations in cancer. Several studies suggest that aberrant methylation occurs in a tumour type-specific manner. However, large-scale analysis of candidate genes has so far been hampered by the lack of high throughput assays for methylation detection. We have developed the first microarray-based technique which allows genome-wide assessment of selected CpG dinucleotides as well as quantification of methylation at each site. Several hundred CpG sites were screened in 76 samples from four different human tumour types and corresponding healthy controls. Discriminative CpG dinucleotides were identified for different tissue type distinctions and used to predict the tumour class of as yet unknown samples with high accuracy using machine learning techniques. Some CpG dinucleotides correlate with progression to malignancy, whereas others are methylated in a tissue-specific manner independent of malignancy. Our results demonstrate that genome-wide analysis of methylation patterns combined with supervised and unsupervised machine learning techniques constitute a powerful novel tool to classify human cancers.

Algorithms↗

PubMed2002

Transscleral optical coherence tomography: a new imaging method for the anterior segment of the eye.

OBJECTIVE: To present a new imaging method for the anterior segment of the eye. METHODS: Transscleral optical coherence tomographic images were generated in healthy volunteers using a slitlamp-adapted prototype equipped with a superluminescence diode with an infrared wavelength of 1310 nm. RESULTS: The optical coherence tomographic system used allowed penetration of human sclera in vivo and high-resolution, cross-sectional imaging of the anterior chamber angle and the ciliary body. CONCLUSION: The 1310-nm optical coherence tomographic image shows a significant potential as a noninvasive diagnostic tool for the anterior segment of the eye.

Anatomy, Cross-Sectional↗