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W G Thies

Publications and source records attributed to W G Thies.

10 recordsLinked to original sources
PubMed2001

Gene expression patterns associated with the metastatic phenotype in rodent and human tumors.

Using subtractive technology, we have generated metastasis-associated gene expression profiles for rat mammary and pancreatic adenocarcinomas. Several genes whose expression is thought to be related to tumor progression such as c-Met, urokinase-type plasminogen activator receptor, ezrin, HMG-1, oncomodulin, cathepsin, and caveolin were thereby isolated. Half of the metastasis-associated clones showed no significant homology to genes with known function. Notably, several of the metastasis-associated clones were also expressed in metastatic lines but not in nonmetastatic lines of other tumor models. Furthermore, in situ hybridization using selected clones documents the relevance of these results for human cancer because strong expression in tumor cells including metastases was detected in human colorectal cancer samples and, to a lesser extent, in mammary cancer samples. These data support the concept that tumors express a "metastatic program" of genes.

Animals↗
PubMed1997

A high throughput screening for rarely transcribed differentially expressed genes.

A novel method combining elements of suppression subtractive hybridization with high throughput differential screening permits the efficient and rapid cloning of rarely transcribed differentially expressed genes. The experimental strategy virtually excludes the possibility of isolating false positive clones. The potential of the method is demonstrated by the isolation of 625 differentially expressed cDNAs from the metastatic adenocarcinoma cell line Bsp73-ASML when subtracted from its non-metastatic counterpart Bsp73-1AS. Northern analysis of 72 randomly selected clones demonstrated that 68 were differentially expressed with respect to Bsp73-ASML, indicating a true positive rate of 94%. Additionally, a large proportion of these clones represented rare transcripts as determined by the exposure time required to detect a signal. Sequence data indicated that of the 625 clones obtained, 92 clones scored perfect or near perfect matches with already known genes. Two hundred and eighty one clones scored between 60 and 95% homology to known human and mouse genes, whereas 252 clones scored no match with any sequences in the public databases. The method we describe is ideally suited whenever subtle changes in gene expression profiles need to be determined.

Adenocarcinoma↗
PubMed1997

Comparative in-vivo and in-vitro 99Mo-time-differential-perturbed-angular-correlation studies on the nitrogenase MoFe protein and on other Mo species of different N2-fixing bacteria.

Klebsiella pneumoniae, Azotobacter vinelandii and Rhodobacter capsulatus were cultivated in media containing 99MoO4(2-) . The distribution of 99Mo in cells grown under conditions of repression and derepression of nitrogenase synthesis, was investigated by anion-exchange (DEAE-Sephacel) chromatography. Cells of K. pneumoniae took up MoO4(2-) only under conditions of derepression of nitrogenase thus serving the formation of the FeMo cofactor of the MoFe protein (Kp1) as the predominant Mo-containing species. In the case of A. vinelandii, under diazotrophic growth conditions, molybdenum was preferably incorporated into the nitrogenase MoFe protein (Av1). However, if excess amounts of molybdate were present in the medium, molybdenum was also bound to the Mo-storage protein. In the presence of 20 mM NH4+, conditions which completely repress nitrogenase formation, molybdenum accumulated in the Mo-storage protein exclusively. This protein proved to be unstable towards DEAE-Sephacel, apparently releasing all the molybdenum in form of MoO4(2-) during the fractionation procedure. R. capsulatus contained, in addition to the MoFe protein (Rc1), significant amounts of other not-yet-identified Mo species, which partially are formed under conditions of both, repression and derepression of nitrogenase. The Mo centers of all these compounds were characterized by measuring the nuclear quadrupole interaction of the process 99Mo(beta-)99Tc using time differential perturbed angular correlation spectroscopy. The quadrupole coupling constant (v(Q)) determined for the Mo center in MoFe proteins was consistently in the range 66-81 MHz. The values of the coupling constants determined with intact cells and with the isolated, partially purified, MoFe proteins were in very good agreement. For the Mo-storage protein of A. vinelandii, a quadrupole coupling constant of approximately 180 MHz was determined by measurements performed with nitrogenase-repressed cells as well as with gel-filtered cell-free extracts. Our work proves that the relevant study of hyperfine interactions allows the identification of the MoFe protein and also other Mo proteins in vivo as well as in vitro.

Azotobacter vinelandii↗
PubMed1992

X-ray solution scattering reveals conformational changes upon iron uptake in lactoferrin, serum and ovo-transferrins.

X-ray solution scattering has been used for studying the structural changes that take place upon uptake and release of iron from serum and chicken ovo-transferrin and human lactoferrin. In the case of chicken ovo-transferrin, data have been obtained for both the intact protein and the isolated N and C-lobes with and without iron. These studies reveal that both lobes undergo a change that is consistent with an opening of the inter-domain cleft when iron is removed from the protein. We suggest that the conformational change of the protein increases the specificity of receptor binding and that the closed configuration of the iron-loaded protein is one, or perhaps the, decisive step in the mechanism for receptor-mediated endocytosis.

Animals↗
PubMed1992

Influence of protein dynamics on the metal-sites of ovotransferrin.

Using the perturbed angular correlations (PAC) technique, the formation of hafnium-ovotransferrin complexes has been studied. Two binding configurations at each of the two specific binding-sites of the protein have been observed. They are characterized by well-defined electric quadrupole frequencies. Information about the dynamics of the protein was derived from temperature dependent measurements of the relaxation constant. The well-resolved spectra taken with fast BaF2-detectors allow a precise determination of the relaxation behaviour of the protein. The results are compared with the predictions from a hydrodynamic model for the reorientation of macromolecules. Thus the hydrodynamic volume of ovotransferrin and its N-terminal half-molecule were determined. The ovotransferrin volume is in agreement with a value derived for human serum transferrin from small angle neutron scattering. From experiments with immobilized protein material there is evidence for internal protein dynamics which is probed by the Hf-ion bound to the specific metal-sites.

Animals↗
PubMed1987

TDPAC studies of the metal-binding sites in serum transferrin: comparison between 181Hf-labeled human- and rat-serum transferrin.

The binding of hafnium to human serum transferrin was studied using the time differential perturbed angular correlation (TDPAC-) technique. The samples were prepared in vitro by adding 181Hf-NTA solution to human serum. Two specific electric quadrupole interactions were observed, which correspond to two well-defined binding configurations. Their relative intensities depend on the pH, salt- and hafnium-concentrations, and on the incubation time. The present data may be compared with the results of a previous rat serum study, where the hafnium binding to transferrin behaved rather similarly. Small but significant differences, however, can be deduced from the TDPAC-parameters for these human and rat transferrin species. For either binding configuration, the electric field gradient (EFG) is slightly higher in the case of rat transferrin. The most characteristic difference, however, concerns the asymmetry parameter eta 2 of the second binding configuration, which is about 10% smaller for rat serum transferrin. The TDPAC-technique might be used as a sensitive and reliable analytical method to study the metal-binding sites of different transferrin species.

Animals↗
PubMed1986

In vivo and in vitro studies of hafnium-binding to rat serum transferrin.

The binding of hafnium to rat serum transferrin was studied using the time differential perturbed angular correlation (TDPAC) technique. Hafnium is interesting as a toxic metal binding to transferrin because it behaves metabolically similarly to plutonium. The isotope 181Hf offers favorable access to the TDPAC-method. Samples were prepared in vivo by intravenous injection of Hf-NTA, Hf-citrate, and Hf-oxalate solutions, respectively, into Sprague-Dawley rats and in vitro by adding Hf-NTA solution to fresh rat serum. In both cases two specific electric quadrupole interactions were observed, which correspond to two well-defined binding configurations. They may be attributed to the N-terminal and the C-terminal binding site in the transferrin molecule. The 181Hf-distribution between these two binding states depends on pH, salt and hafnium concentrations, temperature, and incubation time. With a fast TDPAC-setup of four BaF2-detectors a time resolution of about 600 ps could be achieved. The specific binding configurations of 181Hf and the comparatively slow relaxation times lead to spectra of considerable accuracy.

Animals↗
PubMed1985

Association of 239Pu with lysosomes in rat, Syrian hamster, and Chinese hamster liver as studied by carrier-free electrophoresis and electron microscopic autoradiography with 241Pu.

The binding of injected monomeric plutonium in the liver of rats, Syrian hamsters, and Chinese hamsters (species which show profound differences in their ability to eliminate 239Pu from the liver) was investigated by carrier-free electrophoresis using 239Pu and electron microscopic autoradiography with 241Pu. These studies are part of a program designed to obtain a better understanding of the mechanisms of the clearance of transuranium elements from liver of different mammals and man. Between 4 and 9 days after nuclide injection, a clear correlation between the majority of the 239Pu and lysosomal enzymes was observed when the mitochondrial-lysosomal (ML) fraction of the livers was analyzed by carrier-free electrophoresis. In the two hamster species, a second 239Pu peak exists from the beginning and increases with time to comprise 50% of the total radioactivity at later times. During electron microscopic examination 4 days after 241Pu injection, beta tracks were frequently observed over globular structures resembling dense bodies in Chinese hamster liver. They were also observed frequently over chromatin-rich portions of the cell nuclei. These results, together with those from previous density gradient studies, show that lysosomes are the primary deposition site for 239Pu in the liver cytoplasm of these three rodent species. The hypothesis of a morphologic transformation of these lysosomes with time in hamster liver and of rapid bulk exocytosis of the lysosomes in rats are still possible explanations for the extreme differences in the elimination among the three species.

Animals↗
PubMed1984

Comparative studies on the lysosomal association of monomeric 239Pu and 241Am in rat and Chinese hamster liver: analysis with sucrose, metrizamide, and Percoll density gradients of subcellular binding as dependent on time.

The binding of 239Pu and 241Am in the livers of Chinese hamsters and rats was analyzed by centrifugation of a mitochondrial-lysosomal fraction in sucrose, metrizamide, and Percoll density gradients at intervals between 4 and 70 days after nuclide injection. The behavior of 239Pu and 241Am during the centrifugation experiments was very similar. In contrast to the results for rats, the median densities of the nuclide profiles from hamsters decrease with time in hyperosmolar sucrose gradients, as does the nuclide fraction liberated by addition of Triton X-100, and the nuclide profiles do not respond typically to Triton WR 1339 treatment of the animals. The results with nearly iso- osmolar metrizamide gradients, together with those for Percoll, agree with the assumption that there is an initial lysosomal association of the transuranium elements. It was concluded from the results that the major fraction of 239Pu and 241Am remained bound to typical lysosomes in rat liver, whereas those in hamster liver may be transformed into telolysosomes . Possibly, a vesicular biliary transport system for certain heavy metals, for which evidence exists in rat liver, does not occur in Chinese hamster liver.

Americium↗