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Biomedical subjects

M Schweiger

Publications and source records attributed to M Schweiger.

At least 55 records · Page 3Linked to original sources
PubMed1998

Involvement of the Fanconi anemia protein FA-C in repair processes of oxidative DNA damages.

Fanconi anemia (FA) is an autosomal recessive disorder characterized by skeletal abnormalities, pancytopenia and a marked predisposition to cancer. FA cells exhibit chromosomal instability and hypersensitivity towards oxygen and cross-linking agents such as diepoxybutane and mitomycin C. An increased level of reactive oxygen intermediates and an elevation of 8-oxoguanine in FA cells point to a defective oxygen metabolism in FA cells. We investigated the repair activity of oxidatively damaged DNA in lymphoblastoid cells from FA patients of complementation groups A-E. The repair activity for oxidatively damaged DNA was significantly reduced in lymphoblastoid cell lines of complementation groups B-E. Complementation of the FA-C cell line with the wild type FA-C gene restored the repair activity to normal. This indicates that the FA-C protein participates in the repair of oxidatively damaged DNA.

Cell Cycle Proteins↗
PubMed1998

Regulation of RNA polymerase II-dependent transcription by poly(ADP-ribosyl)ation of transcription factors.

Poly(ADP-ribosyl) transferase (ADPRT) is a nuclear protein that modifies proteins by forming and attaching to them poly(ADP-ribose) chains. Poly(ADP-ribosyl)ation represents an event of major importance in perturbed cell nuclei and participates in the regulation of fundamental processes including DNA repair and transcription. Although ADPRT serves as a positive cofactor of transcription, initiation of its catalytic activity may cause repression of RNA polymerase II-dependent transcription. It is demonstrated here that ADPRT-dependent silencing of transcription involves ADP-ribosylation of the TATA-binding protein. This modification occurs only if poly(ADP-ribosyl)ation is initiated before TATA-binding protein has bound to DNA and thereby prevents formation of active transcription complexes. Specific DNA binding of other transcription factors including Yin Yang 1, p53, NFkappaB, Sp1, and CREB but not c-Jun or AP-2 is similarly affected. After assembly of transcription complexes initiation of poly(ADP-ribosyl)ation does not influence DNA binding of transcription factors. Accordingly, if bound to DNA, transcription factors are inaccessible to poly(ADP-ribosyl)ation. Thus, poly(ADP-ribosyl)ation prevents binding of transcription factors to DNA, whereas binding to DNA prevents their modification. Considering its ability to detect DNA strand breaks and stimulate DNA repair, it is proposed that ADPRT serves as a molecular switch between transcription and repair of DNA to avoid expression of damaged genes.

ADP Ribose Transferases↗
PubMed1998

Analysis of the TPA regulatory element in the genomic poly(ADP-ribose) synthetase gene in human leukemia U937 cells.

The human leukemia U937 cells differentiate into monocyte/macrophage-like cells when treated with 12-O-tetradecanoylphorbol-13-acetate (TPA). We observed that during this process, both protein and mRNA levels for PARS markedly decreased in U937 cells. Through deletion analysis of the PARS regulatory gene, we found that the sequence within the first intron region was responsible for the TPA-dependent repression. Electrophoretic mobility shift assays (EMSAs) and Southwestern blot analysis indicate that this element bound specifically to a nuclear protein. TPA treatment abolished the binding of the protein in U937 cells but not in HeLa cells. DNase I footprinting data show that the cis regulatory element is located between residues 328 and 383. We further examined the function of this cis element (BS207) in a basal promoter regulatory reporter construct and found that this cis element (BS207) functions as an enhancer via the binding of an unknown trans-acting factor. TPA treatment diminished the binding activity of the factor in U937 cells, resulting in a decrease in the enhanced activity to the basal level. These results suggest that abolishment of the binding of a special nuclear protein to the first intron of the PARS gene is related to the TPA-responsive downregulation of PARS in U937 cells.

Base Sequence↗
PubMed1998

Enzymic, cysteine-specific ADP-ribosylation in bovine liver mitochondria.

NAD+ glycohydrolase (NADase) and non-enzymic ADP-ribosylation have been thought to be involved in the regulation of mitochondrial Ca2+ fluxes. In this study it was found that several conditions (5 mM nicotinamide, 5 mM 3-aminobenzamide, 2 mM EDTA, 1 mM ATP, 10 mM dithiothreitol) known to strongly inhibit the NADase decreased ADP-ribosylation in bovine liver mitochondrial membranes with [32P]NAD+ as substrate to only a limited extent, if at all. The reaction led to the specific modification of two proteins with apparent molecular masses of approx. 26 and 53 kDa. An excess of added free ADP-ribose diminished the incorporation of label from [32P]NAD+ only slightly. Dithiothreitol inactivated the NADase, whereas ADP-ribosylation was unaffected. At low concentrations (25 microM) ADP-ribosylation was efficient with NAD+, but not ADP-ribose, as substrate. Under these conditions mitochondrial ADP-ribosylation seems to occur as an enzymic reaction rather than a non-enzymic transfer of ADP-ribose previously liberated from NAD+ by NAD+ glycohydrolase. The chemical stability of the protein-ADP-ribose bonds in the mitochondrial membranes indicated that cysteine residues are the predominant acceptors. Moreover, yeast aldehyde dehydrogenase, known to be a substrate for thiol-associated ADP-ribosylation, was efficiently ADP-ribosylated by using the mitochondrial activity and NAD+ as substrate. The modification of a cysteine residue in the aldehyde dehydrogenase was verified by the observation that pretreatment of this acceptor protein with N-ethylmaleimide substantially decreased its modification. It is therefore concluded that bovine liver mitochondria contain a cysteine-specific ADP-ribosyltransferase.

Adenosine Diphosphate Ribose↗
PubMed1998

A novel function of poly(ADP-ribosyl)ation: silencing of RNA polymerase II-dependent transcription.

Poly(ADP-ribosyl) transferase (ADPRT) is a nuclear enzyme that catalyzes the synthesis of ADP-ribose polymers from NAD+ as well as the transfer of these polymers onto acceptor proteins. The predominant acceptor of the poly(ADP-ribose) chains appears to be the enzyme itself. The function of ADPRT is thought to be related to a number of nuclear processes, including DNA repair and transcription. In this study, it was found that polymerase II-dependent transcription in nuclear HeLa extracts was repressed in the presence of NAD+ at concentrations as low as 1 microM. This repression was strictly dependent on the activity of ADPRT and correlated with the auto(ADP-ribosyl)ation of the enzyme. Subsequent degradation of the ADP-ribose polymers by enzymatic activities present in the nuclear extracts restored transcriptional activity. It would appear from these results that poly(ADP-ribosyl)ation represents the key event of the mechanism underlying NAD(+)-dependent silencing of transcription. Importantly, ADPRT- and NAD(+)-dependent silencing was observed only if poly(ADP-ribosyl)ation had taken place before formation of the transcription complex was completed. That is, if the nuclear extract was preincubated for more than 15 min in the presence of template DNA, transcription was rendered entirely insensitive to NAD+. These results suggest that poly(ADP-ribosyl)ation may prevent polymerase II-dependent transcription, but does not interfere with ongoing transcription. Taking into account the known function of ADPRT, this enzyme may facilitate recovery from DNA damage by stimulating DNA repair and silencing transcription.

Enzyme Activation↗
PubMed1998

Overexpression of thioredoxin in Fanconi anemia fibroblasts prevents the cytotoxic and DNA damaging effect of mitomycin C and diepoxybutane.

Adult T cell leukemia derived factor (ADF)/thioredoxin (Trx) is known to be an important intracellular antioxidant involved in a number of redox reactions such as ribonucleotide reductase (RNR) as well as of tyrosinase. Since RNR is a key enzyme of nucleotide metabolism and DNA synthesis, a reduced Trx level would result in reduced enzymatic activity and cause DNA damage. Furthermore, Trx is considered to be an effective regulator of redox sensitive gene expression. The role of Trx in nucleotide metabolism and gene expression may be an explanation for increased chromosomal instability as well as hypersensitivity towards oxygen, ROI and ROI generating agents. The activity of tyrosinase, the key enzyme of melanin biosynthesis, is influenced by the thioredoxin level and by superoxide radicals. Low thioredoxin levels and high superoxide concentrations activate tyrosinase causing hyperpigmentation of the skin. In addition to the observed high superoxide concentration in Fanconi anemia (FA) patients, a low thioredoxin level might be responsible for the hyperpigmentation (café-au-lait spots) in this disease. We observed that overexpression of the thioredoxin cDNA in FA fibroblasts completely abolished the DNA damaging effects of mitomycin C and diepoxybutane and inhibited the constitutive activity of the nuclear factor kappaB (NF-kappaB) in SV40 transformed FA fibroblasts. However, spontaneous chromosomal breakage was not affected.

Antioxidants↗
PubMed1997

Interaction of the transcription factor YY1 with human poly(ADP-ribosyl) transferase.

Poly(ADP-ribosyl) transferase (ADPRT) is a nuclear enzyme that catalyzes the synthesis of ADP-ribose polymers from NAD+ as well as the transfer of these polymers onto acceptor proteins. The function of ADPRT is thought to be related to a number of nuclear processes including DNA repair and transcription. The transcription factor Yin Yang 1 (YY1) is a potent regulator of RNA polymerase II (Pol II)-dependent transcription. In this study Alu-retroposon-associated binding sites for YY1 located in the distal region of the promoter of the human ADPRT gene have been identified suggesting a possible involvement of this protein in the regulation of ADPRT-gene expression. In the presence of the recombinant automodification domain of the ADPRT the formation of specific YY1 complexes, detected in gel-shift experiments, was strongly inhibited, indicating that this domain of the enzyme may interact directly with YY1. In accordance with this result YY1 was specifically precipitated from nuclear extracts by ADPRT immobilized on sepharose. These results suggest a direct ADPRT-YY1 interaction which may be of importance in the regulation of Pol II-dependent transcription. They also indicate that in some human promoters this regulation may be mediated by retroposons of the Alu family.

Binding, Competitive↗
PubMed1997

Identification of bovine liver mitochondrial NAD+ glycohydrolase as ADP-ribosyl cyclase.

The present investigation identifies bovine liver mitochondrial NADase (NAD+ glycohydrolase) as a member of the class of bifunctional ADP-ribosyl cyclases/cyclic ADP-ribose hydrolases, known to be potential second messenger enzymes. These enzymes catalyse the synthesis and degradation of cyclic ADP-ribose, a potent intracellular calcium-mobilizing agent. The mitochondrial enzyme utilized the NAD+ analogues nicotinamide guanine dinucleotide (NGD+) and nicotinamide hypoxanthine dinucleotide (NHD+) to form fluorescent cyclic purine nucleoside diphosphoriboses. ADP-ribosyl cyclase activity was also demonstrated using 32P-labelled NAD+ as substrate. The identity of NADase and ADP-ribosyl cyclase was supported by their co-migration in SDS/polyacrylamide gels. Cyclase activity was visualized directly within the gel by detecting the formation of fluorescent cyclic IDP-ribose from NHD+. The enzyme catalysed the hydrolysis of cyclic ADP-ribose to ADP-ribose. Moreover, in the presence of nicotinamide and cyclic ADP-ribose the enzyme synthesized NAD+. Both the ADP-ribosyl cyclase and NADase activities of the enzyme were strongly inhibited by reducing agents. Treatment of the NADase with dithiothreitol caused the apparent inactivation of the enzyme. Subsequent removal of the reducing agent and addition of oxidized glutathione led to a partial recovery of enzymic activity. The results support a model for pro-oxidant-induced calcium release from mitochondria involving cyclic ADP-ribose as a specific messenger, rather than the non-enzymic modification of proteins by ADP-ribose.

ADP-ribosyl Cyclase↗
PubMed1997

Image reconstruction in optical tomography.

Optical tomography is a new medical imaging modality that is at the threshold of realization. A large amount of clinical work has shown the very real benefits that such a method could provide. At the same time a considerable effort has been put into theoretical studies of its probable success. At present there exist gaps between these two realms. In this paper we review some general approaches to inverse problems to set the context for optical tomography, defining both the terms forward problem and inverse problem. An essential requirement is to treat the problem in a nonlinear fashion, by using an iterative method. This in turn requires a convenient method of evaluating the forward problem, and its derivatives and variance. Photon transport models are described for obtaining analytical and numerical solutions for the most commonly used ones are reviewed. The inverse problem is approached by classical gradient-based solution methods. In order to develop practical implementations of these methods, we discuss the important topic of photon measurement density functions, which represent the derivative of the forward problem. We show some results that represent the most complex and realistic simulations of optical tomography yet developed. We suggest, in particular, that both time-resolved, and intensity-modulated systems can reconstruct variations in both optical absorption and scattering, but that unmodulated, non-time-resolved systems are prone to severe artefact. We believe that optical tomography reconstruction methods can now be reliably applied to a wide variety of real clinical data. The expected resolution of the method is poor, meaning that it is unlikely that the type of high-resolution images seen in computed tomography or medical resonance imaging can ever be obtained. Nevertheless we strongly expect the functional nature of these images to have a high degree of clinical significance.

Image Processing, Computer-Assisted↗
PubMed1997

Protein-protein interaction of the human poly(ADP-ribosyl)transferase depends on the functional state of the enzyme.

Poly(ADP-ribosyl)transferase (pADPRT) is a nuclear protein which catalyzes the polymerization of ADP-ribose using NAD+ as substrate, as well as the transfer of ADP-ribose polymers to itself and other protein acceptors. The catalytic activity of pADPRT strictly depends on the presence of DNA single-strand breaks. In this report, protein-protein interaction of pADPRT was found to depend on both the extent of automodification with poly(ADP-ribose) and the presence of DNA. Specific binding of radiolabeled pADPRT to transblotted proteins was first tested in blot overlay experiments. For radiolabeling, use was made of the ability of the enzyme to incorporate [32P]ADP-ribose from [32P]NAD+. Varying the concentration of NAD+, two different forms of automodified pADPRT were obtained: oligo(ADP-ribosyl)ated pADPRT with less than 20 ADP-ribose units per chain, and poly(ADP-ribosyl)ated pADPRT with polymer lengths of up to 200 ADP-ribose residues. Interaction of these probes with transblotted HeLa nuclear extracts, purified histones, and distinct regions of recombinant pADPRT was investigated. While the oligo(ADP-ribosyl)ated enzyme associated preferentially with transblotted purified histones, or pADPRT present in HeLa nuclear extracts, poly(ADP-ribosyl)ated pADPRT bound to a variety of transblotted proteins in the nuclear extracts. In the presence of DNA, both the oligo- and the poly(ADP-ribosyl)ated enzymes bound to the transblotted recombinant zinc finger domain of pADPRT even at high salt concentrations. In the absence of DNA, the transblotted automodification domain of pADPRT appeared to be the region involved in self-association. In another set of experiments, unmodified or poly(ADP-ribosyl)ated pADPRT was immobilized on Sepharose. Affinity precipitation of recombinant pADPRT domains confirmed the specific interaction of pADPRT with its zinc finger region and the automodification domain, whereas no interaction was observed with the NAD+ binding domain. Affinity precipitation of HeLa nuclear extracts with poly(ADP-ribosyl)ated pADPRT-Sepharose led to the enrichment of a number of proteins, whereas nuclear proteins bound to the unmodified pADPRT-Sepharose in a smaller extent. The results suggest that protein-protein interaction of the human pADPRT is governed by its functional state.

Adenosine Diphosphate Ribose↗
PubMed1997

Characterization of hydrosoluble and detergent-solubilized forms of mitochondrial NAD+ glycohydrolase from bovine liver.

Treatment of isolated bovine liver mitochondria with either detergents or a crude pancreatic lipase, steapsin, resulted in solubilization of NAD+ glycohydrolase (NADase) activity. The two forms of this enzyme can be visualized directly in SDS-polyacrylamide gels (PAGs) by a fluorescence assay utilizing 1,N6-etheno-NAD+ (epsilon-NAD+) as substrate. Only a slight difference of about 2,000 in the apparent molecular masses was detected. Values of 28,000 and 30,000 for the steapsin- and detergent-solubilized enzyme, respectively, were estimated. The catalytic properties as well as the dependence on temperature, pH, and ionic strength were found to be similar for both forms of the enzyme. One important difference regarding their sensitivity against bivalent metal ions was observed. While the detergent-solubilized NADase was activated in the presence of, for example, Zn++, and inhibited by EDTA, the truncated enzyme seemed to be unaffected under these conditions.

Animals↗
PubMed1997

The role of oxygen metabolism for the pathological phenotype of Fanconi anemia.

The molecular defect of the hereditary disease Fanconi anemia (FA) remains unknown. The two theoretical possibilities are (1) an impaired DNA crosslink-repair system or (2) a disturbed oxygen metabolism either by overproduction of reactive oxygen intermediates (ROI) or by diminished detoxification of ROI. In order to gain further insight into the molecular mechanism of this disease, we have determined the repair capacity of FA cells challenged by crosslinking agents and have analyzed diverse biological systems that are involved in oxygen metabolism. We have tested normal and FA cells for oxygen consumption and for the activity of the antioxidant phospholipid-hydroperoxide-glutathione-peroxidase (PHGPx). FA cells show a reduced oxygen consumption and an increased PHGPx activity. Since spontaneous and induced chromosomal instability is a main cellular feature of FA, we have analyzed the redox state of cells and the effect of cytochrome P-450 (Cyt P-450) inhibitors and inducers on chromosomal breaks and micronuclei production. Our results indicate that Cyt P-450 enzymes, especially Cyt P-450 1A2, play a crucial role in radical metabolism in FA cells. Furthermore, we have determined NF-kappa B activity in untransformed cells and in SV40-transformed cells by gel shift experiments. NF-kappa B is a multiunit transcription factor that is known to be induced by ROI and that activates the expression of various genes involved in cellular responses to stress. NF-kappa B is constitutively induced in SV40-transformed FA cells probably as a consequence of an increased ROI level. Our results suggest that enzymatic defects in oxygen metabolism mediate the FA phenotype via impaired reactivity with ROI. Cyt P-450 1A2 appears to be a good candidate for the defective enzyme, even though no differences have been measured in the activity of this enzyme in FA and control fibroblasts in pilot experiments.

Cell Line↗
PubMed1997

The finite-element method for the propagation of light in scattering media: frequency domain case.

A frequency domain light transport model to simulate the transillumination of a scattering object with radio frequency intensity modulated light is presented. The model is based on the diffusion approximation to the radiative transfer equation and uses a finite-element model to allow for complex geometries and an inhomogeneous distribution of absorption and scattering. It calculates the complex photon density within the object and the complex exitance on the boundary of the object. The model is validated against an analytic Green's function model for a circular geometry in the homogeneous case, and its accuracy is investigated for a range of mesh resolutions, optical parameters, and modulation frequencies.

Biophysical Phenomena↗
PubMed1997

Routes of asbestos exposure and the development of mesothelioma in an English region.

OBJECTIVES: To investigate the contribution of exposure to asbestos through different routes in the development of mesothelioma. METHODS: Case-control study. 185 confirmed cases of mesothelioma and 160 controls were identified, when death had occurred between 1979 and 1991 in four health districts in Yorkshire. The surviving relatives were interviewed to ascertain lifetime exposure to asbestos. Adjusted odds ratios (ORs) of exposure to asbestos (through occupational, paraoccupational, and residential routes) were calculated for cases and were compared with controls. RESULTS: Likely or possible occupational exposure to asbestos was more common in cases than in controls (OR 5.6, 95% confidence interval (95% CI) 3.1 to 10.1). After excluding those with likely or possible occupational exposure, likely or possible paraoccupational exposure was more common in cases than controls (OR 5.8, 95% CI 1.8 to 19.2). Only six cases of mesothelioma were identified as being solely exposed to asbestos through their residence, compared with nine controls. The OR for residential exposure to asbestos varied between 1.5 and 6.6, depending on which potential industrial sources were included, but the 95% CIs were so wide that slightly reduced or greatly increased odds comparing cases with controls could not be excluded. CONCLUSION: Study results support previous evidence that occupational and paraoccupational exposure to asbestos is associated with developing mesothelioma. Despite a rigorous search, purely residential exposure seemed to account for only 3% of identified cases. No firm conclusion can be drawn about the risks from residential exposure alone, as many of the study subjects could also have been occupationally or paraoccupationally exposed to asbestos.

Adult↗
PubMed1997

Bovine liver mitochondrial NAD+ glycohydrolase. Relationship to ADP-ribosylation and calcium fluxes.

Mitochondrial NAD+ glycohydrolase (NADase) has been proposed to be required for (nonenzymatic) ADP-ribosylation and subsequent activation of a Ca2+ release pathway. In our studies it has been found that several agents including nicotinamide, dithiothreitol, and EDTA exert no or little effect on ADP-ribosylation in isolated bovine liver mitochondria, while strongly inhibiting the NADase. The NADase did, however, catalyze the formation of cyclic purine nucleoside diphosphoriboses (similar to cyclic ADP-ribose) from NAD+ analogs. It appears possible, therefore, that this enzyme may be involved in the regulation of mitochondrial Ca2+ fluxes by forming a potent Ca(2+)-mobilizing agent, rather than by providing the substrate for non-enzymatic ADP-ribosylation.

Adenosine Diphosphate Ribose↗