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Biomedical subjects

W Waldeck

Publications and source records attributed to W Waldeck.

At least 19 recordsLinked to original sources
PubMed2006

DNA damaging capability of hematoporphyrin towards DNAs of various accessibilities.

In this work we wanted to verify that photoactivation of DNA-non-binding porphyrin derivative hematoporphyrin IX (Hp) is able to induce damages in DNAs of various accessibilities such as B-conformation and superhelical isolated DNA, nucleoprotein complex and intracellular DNAs. It was found that photodynamic reaction of Hp results significant changes in thermal stability of isolated T7 DNA and induces single strand breaks in supercoiled Bluescript plasmid isolated from Escherichia coli cells. As optical melting measurements revealed, the irradiation of photosensitized T7 nucleoprotein complex leads to a destabilization of the protein capsid. The photodynamic reaction affected both the protein structure and DNA-protein interaction, however, the parameters corresponding to the DNA denaturation are not influenced. The accumulation of Hp in HeLa cells was followed by laser scanning confocal microscopy. The picture received is typical for lipophilic dyes. When Hp loaded cells were irradiated, a reduction of viability could be observed in a concentration and a light dose dependent manner; 12microM porphyrin induced almost complete cell killing after 30min irradiation. After similar treatment, alkaline agarose gel electrophoresis of isolated nuclear DNA did not show the presence of single strand breaks. The alkaline comet assay also failed to demonstrate any DNA damage in HeLa cells. We also considered the possibility of the generation of damages in intracellular SV40 DNA. According to the electropherograms there was no difference between the patterns of DNAs from treated and control samples.

DNA↗
PubMed2005

Development and characterization of drug delivery systems for targeting mammalian cells and tissues: a review.

Recent advances in the development of diagnostics and therapeutics in the fields of recombinant biochemistry, solid phase peptide synthesis as well as in galenical research have resulted in highly specific and efficient components. Presently, millions of patients can profit from these new therapeutic modalities. The application of an effective anti-tumor dose of drugs can lead to marked toxicity in patients. Therefore, safe and efficient possibilities to transport these compounds to the target are of outmost importance. The importance of drug delivery is pivotal in the wide area of pharmacological research. However, until now, this issue is still to be solved. The main goal of every drug delivery system is the delivery of a precise amount of a drug at a pre-programmed rate to the desired location in order to achieve the necessary drug concentration in the targeted organ for effective treatment. The key problem still remains the achievement of curative doses in a pharmacologically active state in the desired target while avoiding side effects. Although respectable advances can be recognized in this field, the currently applied mechanisms for the transport of therapeutic molecules across biological membranes still remain far from being efficient. Helper molecules could improve delivery to desired target sites. Presently, a number of efforts are made and a huge spectrum of biochemical, biological, medical, pharmaceutical and physical possibilities are arising. However, the design and development of successful therapies based on this technology still remains a great challenge.

Animals↗
PubMed2004

Characterization of simian virus 40 on its infectious entry pathway in cells using fluorescence correlation spectroscopy.

SV40 (simian virus 40) is a double-stranded DNA virus and is strongly oncogenic in experimental animals. SV40 enters cells by atypical endocytosis mediated by caveolae, transporting the virus to its usual destination, namely the endoplasmic reticulum. The cellular mechanisms of capsid disassembly (uncoating) and deliverance of the viral genome into the cellular nucleus remain unknown. Here, we study (i) the formation of caveolae after viral infection and the diffusion of caveosome vesicles in the cytoplasm and (ii) the capsid disassembly and the mobility of the viral genome on its way to the nucleus, using fluorescence correlation spectroscopy. To follow the viral genome and capsids separately, the histone components of SV40 minichromosomes were labelled with enhanced yellow fluorescent protein and the capsid was labelled with a fluorescent red dye, Alexa568. We characterized the diffusion of caveosomes, the capsid disassembly process in the cytoplasm and the mobility of the viral genome in the nucleus, using two kinds of permissive cells.

Animals↗
PubMed2002

Preferential radiosensitization in p53-mutated human tumour cell lines by pentoxifylline-mediated disruption of the G2/M checkpoint control.

PURPOSE: There is evidence that the duration of the G2/M delay following irradiation is correlated with cell survival. We studied the radiosensitizing potential of pentoxifylline (PTX) and the PTX-mediated modulation of cell-cycle progression dependent on the p53 status of various human tumour cell lines. MATERIALS AND METHODS: The cellular radiosensitivity of human MCF-7 (wild-type p53) and HT-29 (p53-defective) tumour cells, which were exposed to PTX (2 mM) immediately after gamma-irradiation was determined by colony forming assay. The influence on cell cycle progression after irradiation (6 Gy) was assessed by flow cytometric analysis using p53 wild-type MCF-7 and HPR600 cells, and p53-defective HT-29 and WiDr cells. RESULTS: Clonogenic survival assays up to 8 Gy demonstrated that p53-defective HT-29 cells (sensitizer enhancement ratio [SER]=1.54) were sensitized by PTX (2 mM) to a significantly higher degree than p53 wild-type MCF-7 (SER=1.14) cells. Exposure of irradiated (6 Gy) cells to PTX (2 mM) resulted in abrogation of the radiation-induced G2/M arrest in the p53-defective HT-29 and WiDr cells, whereas the p53 wild-type-expressing MCF-7 and HPR600 cells showed less significant impairment of the G2/M checkpoint. In HT-29 cells, the rate of transition into mitosis was even higher than in the sham-treated control cells. G2/M abrogation was accompanied by an increase of apoptosis only in HPR600 cells. CONCLUSIONS: Since PTX was less effective in cells expressing intact p53, the application of PTX suggests a promising strategy of pharmacological disruption of the G2/M checkpoint control by which preferentially radiation-resistant tumours with defective p53 function might be rendered more sensitive to ionizing radiation.

Cell Cycle↗
PubMed2000

Anomalous diffusion of fluorescent probes inside living cell nuclei investigated by spatially-resolved fluorescence correlation spectroscopy.

We have investigated spatial variations of the diffusion behavior of the green fluorescent protein mutant EGFP (F64L/S65T) and of the EGFP-beta-galactosidase fusion protein in living cells with fluorescence correlation spectroscopy. Our fluorescence correlation spectroscopy device, in connection with a precision x-y translation stage, provides submicron spatial resolution and a detection volume smaller than a femtoliter. The fluorescence fluctuations in cell lines expressing EGFP are caused by molecular diffusion as well as a possible internal and a pH-dependent external protonation process of the EGFP chromophore. The latter processes result in two apparent nonfluorescent states that have to be taken into account when evaluating the fluorescence correlation spectroscopy data. The diffusional contribution deviates from ideal behavior and depends on the position in the cell. The fluorescence correlation spectroscopy data can either be evaluated as a two component model with one fraction of the molecules undergoing free Brownian motion with a diffusion coefficient approximately five times smaller than in aqueous solution, and another fraction diffusing one or two orders of magnitude slower. This latter component is especially noticeable in the nuclei. Alternatively, we can fit the data to an anomalous diffusion model where the time dependence of the diffusion serves as a measure for the degree of obstruction, which is large especially in nuclei. Possible mechanisms for this long tail behavior include corralling, immobile obstacles, and binding with a broad distribution of binding affinities. The results are consistent with recent numerical models of the chromosome territory structure in the cell nucleus.

Animals↗
PubMed2000

Salt-dependent compaction of di- and trinucleosomes studied by small-angle neutron scattering.

Using small-angle neutron scattering (SANS), we have measured the salt-dependent static structure factor of di- and trinucleosomes from chicken erythrocytes and from COS-7 cells. We also determined the sedimentation coefficients of these dinucleosomes and dinucleosomes reconstituted on a 416-bp DNA containing two nucleosome positioning sequences of the 5S rDNA of Lytechinus variegatus at low and high salt concentrations. The internucleosomal distance d was calculated by simulation as well as Fourier back-transformation of the SANS curves and by hydrodynamic simulation of sedimentation coefficients. Nucleosome dimers from chicken erythrocyte chromatin show a decrease in d from approximately 220 A at 5 mM NaCl to 150 A at 100 mM NaCl. For dinucleosomes from COS-7 chromatin, d decreases from 180 A at 5 mM to 140 A at 100 mM NaCl concentration. Our measurements on trinucleosomes are compatible with a compaction through two different mechanisms, depending on the salt concentration. Between 0 and 20 mM NaCl, the internucleosomal distance between adjacent nucleosomes remains constant, whereas the angle of the DNA strands entering and leaving the central nucleosome decreases. Above 20 mM NaCl, the adjacent nucleosomes approach each other, similar to the compaction of dinucleosomes. The internucleosomal distance of 140-150 A at 100 mM NaCl is in agreement with distances measured by scanning force microscopy and electron microscopy on long chromatin filaments.

Animals↗
PubMed1997

Salt effects on the structure and internal dynamics of superhelical DNAs studied by light scattering and Brownian dynamics.

Using laser light scattering, we have measured the static and dynamic structure factor of two different superhelical DNAs, p1868 (1868 bp) and simian virus 40 (SV40) (5243 bp), in dilute aqueous solution at salt concentrations between 1 mM and 3 M NaCl. For both DNA molecules, Brownian dynamics (BD) simulations were also performed, using a previously described model. A Fourier mode decomposition procedure was used to compute theoretical light scattering autocorrelation functions (ACFs) from the BD trajectories. Both measured and computed autocorrelation functions were then subjected to the same multiexponential decomposition procedure. Simulated and measured relaxation times as a function of scattering angle were in very good agreement. Similarly, computed and measured static structure factors and radii of gyration agreed within experimental error. One main result of this study is that the amplitudes of the fast-relaxing component in the ACF show a peak at 1 M salt concentration. This nonmonotonic behavior might be caused by an initial increase in the amplitudes of internal motions due to diminishing long-range electrostatic repulsions, followed by a decrease at higher salt concentration due to a compaction of the structure.

Biophysical Phenomena↗
PubMed1991

Topological properties of bovine papillomavirus type 1 (BPV-1) DNA in episomal nucleoprotein complexes: a model system for chromatin organization in higher eukaryotes.

Sedimentation analysis of isolated episomal bovine papillomavirus type 1 (BPV-1) nucleoprotein complexes in sucrose gradients and subsequent separation of the purified DNA in chloroquine gels revealed different classes of molecules, varying in their degree of superhelicity. Since torsionally stressed DNA favors the adoption of secondary structures, we employed the single-strand-specific S1 nuclease to detect such structural alterations in both naked DNA and native chromatin. Direct examination of nuclease digestion products in chloroquine gels showed that neither the naked DNA nor the BPV-1 nucleoprotein complexes in isolated nuclei were cleaved randomly by the enzyme. Instead, there was a strict dependence on nuclease susceptibility and the degree of supercoiling, strongly suggesting that the structural features detected by S1 nuclease are due to the occurrence of torsionally stressed viral chromatin. Mapping analysis using the indirect end-labeling method demonstrated an S1-nuclease cleavage site adjacent to 20 homopurine residues known to be hypersensitive to S1 attack. Furthermore, direct methylation experiments with viral chromatin in isolated nuclei indicated that only circular, covalently closed nucleoprotein complexes served as substrate, whereas linearized BPV-1 chromatin was not susceptible to exogenously added Hhal methylase. This observation raises the possibility that the modulation of topology in nucleosomally organized DNA might also play a role in eukaryotic DNA methylation.

Animals↗
PubMed1990

Antiviral xanthate causes conformational changes in simian virus 40 DNA and chromatin.

Xanthates possess a wide antiviral and antitumoral spectrum. Treatment of simian virus 40 (SV40)-infected cells with tricyclo-decan-9-yl-xanthate (D609) reduces transcription and replication. Concomitantly, we observed condensation of the SV40 minichromosomes after addition of D609 to the tissue culture. The structural change of the viral chromatin was measured as an increase in sedimentation behavior from 95-75 to 180-150 Svedberg units. A modulation of the DNA structure was detected in parallel, resulting in an increase in the topological linking number of the SV40 DNA by two superhelical turns above the average superhelicity in the infected cell. Therefore, enzyme activities responsible for topological changes (topoisomerases) were examined and an inhibition of topoisomerase I by more than 1,000-fold was detected.

Animals↗
PubMed1988

Topoisomerase II inhibitors influence simian virus 40 chromatin structure in vivo accompanied with inhibition of replication, transcription and changes in DNA supercoiling.

The association of topoisomerase II enzymes with papovavirus-chromatin has been described recently. We used cells infected with simian virus 40 (SV40) to investigate the in vivo effect of the topoisomerase II inhibitors nalidixic acid and novobiocin on the viral chromatin template. Blocking of topoisomerases inhibits DNA and RNA synthesis. The block with the inhibitors resulted in a conversion of a 95- to 75-svedberg chromatin to a 180- to 150-svedberg component accompanied with a variation in the topological linking number of the DNA. The condensed chromatin fractions (180-150 S) from inhibited cells resemble encapsidation intermediates and their DNA has a linking number as the DNA extracted from purified virions. Moreover, the virion DNA has a higher superhelical density compared to intracellular chromatin isolated from untreated cells and argues for a supercoiling activity in the cell.

Animals↗
PubMed1986

Properties of intracellular bovine papillomavirus chromatin.

Episomal nucleoprotein complexes of bovine papillomavirus type 1 (BPV-1) in transformed cells were exposed to DNase I treatment to localize hypersensitive regions. Such regions, which are indicative for gene expression, were found within the noncoding part of the genome, coinciding with the origin of replication and the 5' ends of most of the early mRNAs. However, there were also regions of hypersensitivity within the structural genes. These intragenic perturbations of the chromatin structure coincide with regulatory sequences at the DNA level. One of these regions maps in close proximity to a Z-DNA antibody-binding site which is located near the putative BPV-1 enhancer sequence.

Animals↗
PubMed1984

Origin of replication in episomal bovine papilloma virus type 1 DNA isolated from transformed cells.

The origin of replication of bovine papilloma virus type 1 (BPV-1) has been determined by isolating replicative intermediates (RI) of BPV-transformed hamster embryo fibroblasts (HEF-BPV). These RI were treated with single cut restriction enzymes to determine the start-position (origin) of the extending replication eyes using electron microscopic techniques. 'Cairns'-type RI molecules were shown to contain one replication eye in monomeric as well as in dimeric molecules. The position of this eye was localized at 6940 +/- 5% bp in the physical map. In a second set of experiments BPV-1 DNA fragments cloned in pBR322 were tested for transient episomal replication. Transfected cells were harvested after increasing periods of time and screened for replication with isoschizomeric restriction enzymes to differentiate between input and replicated DNA. The part of the BPV genome harboring the replication origin spans the BPV ClaI-C restriction fragment corresponding to the non-coding region of the BPV genome and coincides with the DNase I-hypersensitive control region in the chromatin, isolated from transformed cells.

Animals↗
PubMed1983

Catenation of DNA by eucaryotic topoisomerase II associated with simian virus 40 minichromosomes.

After incubation of purified SV40 minichromosomes with superhelical DNA molecules either of SV40 or plasmid origin, a catenation of monomeric DNA via dimers and multimers to large networks was observed. The catenation reaction was stimulated by the DNA condensing agent spermidine with ATP as an energy donor and was dependent on the presence of magnesium ions. The reaction could be blocked by inhibitors of topoisomerase II such as novobiocin and nalidixic acid. Relaxed covalently closed circular DNA was catenated to networks in the presence of ATP as the energy donor.

Animals↗
PubMed1983

Isolation of episomal bovine papillomavirus chromatin and identification of a DNase I-hypersensitive region.

The investigation of papillomavirus chromatin has been hampered by the unavailability of a tissue culture system for vegetative growth of these viruses. We have used, therefore, bovine papillomavirus type 1-transformed hamster embryo fibroblasts containing 200 to 250 episomal genome equivalents per cell as a source of viral chromatin. The selectively isolated chromatin was shown to be slightly larger (80S) than the mature simian virus 40 chromatin, which was cosedimented in a sucrose density gradient. Both Fo I and Fo II were present in the bovine papillomavirus type 1 chromatin. A fast-sedimenting fraction, whose structure is still unknown, also contained oligomeric bovine papillomavirus type 1 DNA. By in situ DNase digestion of isolated nuclei and subsequent cleavage of the bovine papillomavirus type 1 DNA with various restriction endonucleases, a major DNase-hypersensitive region was detected in the chromatin. This region, comprising approximately 320 base pairs, is located between the relative physical map positions 0.88 and 0.92.

Animals↗
PubMed1982

In vitro formation of multimeric DNA structures mediated by purified simian virus 40 chromatin.

Simian virus 40 chromatin was incubated after purification by sucrose density-gradient centrifugation with various circular double-stranded DNA substrates. Monomeric rings were converted in the presence of Mg2+ to structures possessing a higher degree of complexity. Dimeric catenanes, as well as multimeric linear structures and concatemers, were generated, indicating that recombination events had occurred in vitro involving covalent linkage between different DNA molecules. Furthermore, apparently fused dimeric rings were observed. Their structures suggest that they may be recombination intermediates such as those described in a prokaryotic system [Potter, H. & Dressler, D. (1976) Proc. Natl. Acad. Sci. USA 73, 3000-3004]. Recombination did not take place between heterologous DNA substrates, as exclusively homologous multimeric DNA structures were observed.

Bacteriocin Plasmids↗
PubMed1981

Papovavirus chromatin associated cellular endonuclease which introduces one double-strand cut in superhelical deoxyribonucleic acid.

Nuclear extracts from SV40-infected CV-1 monkey kidney cells and from polyoma-infected 3T3 mouse cells contain an endonucleolytic activity which cleaves circular viral DNA within the chromatin to full-length linear rods [Waldeck, W., Föhring, B., Chowdhury, K., Gruss, P., & Sauer, G, (1978) Proc. Natl. Acad. Sci. U.S.A. 75, 5964-5968; Scott, W. A., & Wigmore, D. J. (1978) Cell (Cambridge, Mass.) 15, 1511-1518]. Sedimentation of the nuclear extracts through sucrose density gradients revealed a preferential binding of the endonuclease to the viral chromatin. Deproteinized exogenous covalently closed superhelical DNA substrates such as SV40 and polyoma as well as Col E1 and PM2 DNAs were linearized by the endonuclease by introduction of one double-strand break per molecule. The reaction products, FOIII unit length rods, were shown to be devoid of single-strand nicks by electrophoresis in denaturing agarose gels. The double-strand break was randomly located within the various substrates since redigestion of the FOIII with single-cut restriction endonucleases failed to generate discrete pairs of reaction products. Neither linear double-stranded nor nicked circular FOII DNA structures were accepted as substrates. The endonucleolytic activity does not require the presence of ATP but is sensitive to EDTA. The enzyme activity is of cellular origin since nuclear extracts from uninfected CV-1 cells converted exogenous superhelical DNA to FOIII structures with the same properties as those described above. The biological properties of the endonuclease are discussed in the light of its possible function in permitting genetic exchange between different circular genomes. Further, it may play an essential role late during the replication of papovavirus DNA when the catenated daughter molecules are liberated from each other by an as yet unidentified mechanism.

Animals↗