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Biomedical subjects

E Spiess

Publications and source records attributed to E Spiess.

At least 55 records · Page 3Linked to original sources
PubMed1987

High sensitivity immunolocalization of double and single-stranded DNA by a monoclonal antibody.

A monoclonal antibody (AK 30-10) is described which specifically reacts with DNA both in double and single-stranded forms but not with other molecules and structures, including deoxyribonucleotides and RNAs. When used in immunocytochemical experiments on tissue sections and permeabilized cultured cells, this antibody detects DNA-containing structures, even when the DNA is present in very small amounts. Examples of high resolution detection include the DNA present in amplified extrachromosomal nucleoli, chromomeres of lampbrush chromosomes, mitochondria, chloroplasts and mycoplasmal particles. In immunoelectron microscopy using the immunogold technique, the DNA was localized in distinct substructures such as the "fibrillar centers" of nucleoli and certain stromal centers in chloroplasts. The antibody also reacts with DNA of chromatin of living cells, as shown by microinjection into cultured mitotic cells and into nuclei of amphibian oocytes. The potential value and the limitations of immunocytochemical DNA detection are discussed.

Animals↗
PubMed1986

Invasion of human embryonic fibroblast cell aggregates by rat tumor cells of different metastatic capacities.

Aggregates of normal human Wi38 cells are used as a three dimensional substrate to test in vitro the behavior of rat tumor cells which exhibit different invasive and metastatic capabilities in vivo. The invasive but non metastatic tumor cells colonize the Wi38 cell aggregates, invade and destroy them within three days. The non invasive but highly metastatic tumor cells settle in a limited number on the aggregates but show no further activities. Co-cultivation of these tumor cells with cell suspension of single Wi38 cells under aggregation conditions does not hinder the Wi38 cells in forming aggregates. The results show that the invasive process in the metastatic cascade needs more specific reaction partners and host environment than local tumor growth. The conditions of the first process cannot be mimicked by a simple model.

Animals↗
PubMed1986

In vitro motility of BSp73 rat tumor cells with different metastatic ability.

The highly metastatic and nonmetastatic variants of the rat tumor BSp73 have been tested for motility in vitro by a phagokinetic assay. Surprisingly, the nonmetastatic variant is locomotional, whereas the highly metastatic variant exhibited stationary motility only. Soluble factors of normal rat serum and medium conditioned by regenerating normal rat lung, or solid constituents of the extracellular matrix material which was exudated by normal rat lung fibroblasts or bovine cornea endothelial cells as well as collagen type III could not stimulate locomotory ability in these tumor cells. However, by contact to rat lung cells they acquired passive mobility. The significance of these cellular properties for their metastatic behavior is discussed.

Animals↗
PubMed1985

Mouse DNA-cytosine-5-methyltransferase: sequence specificity of the methylation reaction and electron microscopy of enzyme-DNA complexes.

Monoclonal antibodies prepared against DNA methyltransferase from human placenta undergo immune complex formation also with DNA methyltransferase from P815 mouse mastocytoma cells. One of these monoclonal antibodies, M2B10, was used for the immunoaffinity purification of this enzyme. Complexes of the immunoaffinity-purified mouse DNA methyltransferase with DNA were visualized by electron microscopy. DNA methyltransferase was found to be distributed along linearized plasmid DNA with a higher incidence of enzyme molecules at the terminal segments. This binding to strand ends was significantly increased after dG- or dGdC-tailing of the DNA, which is compatible with a preferred binding of the enzyme to single-stranded DNA. Sequence specificity analysis using methyl-sensitive restriction enzymes showed that the mouse DNA methyltransferase transferred methyl groups to the internal cytosines in 5'CCGG and 5'GCGC sequences, however, the external cytosine in 5'CCGG sequences was also methylated.

Animals↗
PubMed1985

Adhesion and spreading characterization of a rat tumor cell system exhibiting different metastatic behavior.

The rat tumor cell system BSp73AS/BSp73ASML was investigated for its structural aspects. The non-metastasizing line BSp73AS has less nuclear atypism than the metastasizing line BSp73ASML. Microvilli are scanty and variable in length and structure; in BSp73ASML they cover the cells densely, are short and homogeneous in size. In vitro adhesion and spreading tests show structurally flexible BSp73AS cells which flatten completely onto substrata. BSp73ASML cells remain spherical and develop only small attachment areas. In vitro aggregation of the BSp73AS cells leads to round, tightly packed aggregates, their outer cell layer is epitheloid BSp73ASML cells form clusters of mostly ball-shaped cells. Surprisingly, as judged from EM images, the intercellular junctions in BSp73ASML cell clusters are qualitatively the same as in BSp73AS cell aggregates. However, the extent of apposition between cells in BSp73AS aggregates is much higher than between cells in the BSp73ASML clusters. BSp73ASML cells lack the ability to vary their shape. This is the prominent difference revealed by these tests between the two cell lines. Hence, this fact could play an important role in their different metastatic behavior in vivo. We speculate that the ability to vary the cell shape is necessary for expansive growth in the host environment but not for the metastatic spread.

Animals↗
PubMed1984

Highly sensitive sites for guanine-O6 ethylation in rat brain DNA exposed to N-ethyl-N-nitrosourea in vivo.

Brain chromosomal DNA isolated from fetal BDIX-rats 1 h after i.v. administration of the ethylating N-nitroso carcinogen N-ethyl-N-nitrosourea (75 micrograms/g body weight), statistically contained one molecule of O6-ethyl-2'-deoxyguanosine (O6-EtdGuo) per 81 micron of DNA, as determined in enzymatic DNA hydrolysates by competitive radio-immunoassay using a high-affinity anti-(O6-EtdGuo) monoclonal antibody (ER-6). After fragmentation of the DNA by the restriction enzyme AluI (average fragment length, Lav = 0.28 micron = 970 bp; length range, Lr = 1.87-0.02 micron = 6540 - 60 bp), a small (approximately 2%) fraction of DNA enriched in specific polypeptides tightly associated with DNA was separated from the bulk DNA by a glass fiber binding technique. As analyzed by immune electron microscopy, approximately 1% of the DNA molecules in this fraction contained clusters of 2-10 (O6-EtdGuo)-antibody binding sites (ABS). On the cluster-bearing fragments (Lav, 0.85 micron +/- 0.50 micron S.D.; corresponding to 2970 +/- 1760 bp) the average ABS-ABS interspace distance was 110 nm (= 390 bp; range approximately 9-600 nm), indicating a highly non-random distribution of O6-EtdGuo in target cell DNA.

Alkylating Agents↗
PubMed1984

Chromosome analysis of two rat tumor cell lines. Possible role of DMs and HSR in metastasis.

The rat tumor cell lines BSp73AS (AS, non-metastasizing with pronounced adherent capacities) and BSp73ASML (ASML, highly metastasizing with reduced adherent capacities) were cytogenetically investigated. The ASML cell line is reportedly derived from the AS cell line. Both lines exhibited abnormal numerical and structural chromosomal characteristics. The metastasizing ASML cells showed a higher chromosome number (modal number: 62-63) than the nonmetastasizing AS cells (modal number:48). The AS karyotype was characterized by the presence of a large metacentric marker chromosome resulting from a Robertsonian translocation (Rb 6.7). This chromosome is as such absent in ASML cells but perhaps it may be present in these cells with a major part of chromosome 7 being deleted. The most interesting feature of the ASML karyotype was the presence of double minutes (DMs) and a homogeneously staining region (HSR) at the telomeric end of chromosome 6. These were peculiar to the ASML cells, being absent in the AS cells. DMs and HSR are reported to be correlated with the resistance to various drugs and with the acquired virulence of tumor cells through gene amplification. Therefore, we assume that in the metastasizing ASML cell line the DMs and HSR were established through genetic selection and that they are probably related to the acquired metastasizing capacity of these cells.

Animals↗
PubMed1984

Antibodies to the most tightly bound proteins in eukaryotic DNA. Formation of immuno-complexes with 'nuclear matrix' components.

Chromosomal DNA is associated with polypeptides covalently bound to internal DNA ends. Since these polypeptides can only be released from chromosomal DNA by enzymes or other agents hydrolysing phosphodiester bonds they were termed 'the most tightly bound' (MTB) polypeptides in DNA. Antibodies developed against the MTB polypeptides are shown to form immunocomplexes with major 'nuclear matrix' polypeptides as well as with polypeptides which are still associated with 'nuclear matrix' DNA isolated by means of SDS/proteinase K and phenol. Immuno-complex formation is revealed by immunoblotting and by indirect immunofluorescence. Thus, since MTB polypeptides, major 'nuclear matrix' polypeptides and 'nuclear matrix' DNA-associated polypeptides share common antigenic sites they can be considered to be identical or at least closely related. This suggests that a fraction of distinct 'nuclear matrix' polypeptides is either transiently or permanently linked to DNA by covalent bonds. Consistently, isolated eukaryotic 'bulk' DNA is inevitably associated with residual 'nuclear matrix' polypeptides.

Animals↗
PubMed1984

Morphological and behavioral characteristics of two rat tumor cell lines with different metastatic capacities.

The morphological characteristics of two rat tumor cell lines were studied by means of scanning electron microscopy. While the ASML-cells (Ascites, Solid, Metastases, Lung) when spread via the lymphatic vessels form metastases in the lung, the AS-cells (Ascites, Solid) do not form secondary tumors. The ASML-cells do not attach on glass or plastic but grow in suspension; AS-cells, can be cultivated as monolayers. The ASML-cells form loosely packed clusters when they are shaken in a gyratory shaker, AS-cells produce well organized aggregates in which the intercellular contacts are very intensive. When a mixture of cells from both lines is aggregated the two types sort out and the line-specific aggregate or cluster is formed without interaction between the two types. ASML-cells remain spherical when confronted with pieces of the aortic wall and there are no signs of reaction with the underlying endothelial cells. The endothelium is drastically altered when AS-cells are seeded on top of the aortic wall: the tumor cells flatten, penetrate the endothelial layer and induce the endothelial cells to retract. These phenomena can also be seen in confrontations with the mesothelium of the diaphragm. As also cells from aggregates or clusters behave as the single cells we conclude that the newly formed heterotypic contacts are favored over homotypic ones. In addition they have proved to be more intensive.

Animals↗
PubMed1984

High-affinity monoclonal antibodies for the specific recognition and quantification of deoxynucleosides structurally modified by N-nitroso compounds.

The applicability of conventional radiochromatographic procedures to the detection and quantification of specific, carcinogen-induced structural modifications in the DNA of mammalian cells is limited by the necessity of using radioactively labelled agents and by the relatively large amounts of DNA required for analysis of low levels of DNA modification. Recently developed immunoanalytical methods have improved this situation considerably. High-affinity monoclonal antibodies (MAB), in combination with radio- and enzyme-immunoassays, now permit the sensitive detection of alkyldeoxynucleosides in small samples of hydrolysed DNA from tissues and cultured cells exposed previously to non-radioactive (e.g., environmental) alkylating N-nitroso carcinogens. Furthermore, MAB can be used to quantify by direct immunofluorescence (and with the aid of computer-based image analysis of electronically intensified fluorescence signals) specific alkylation products in the DNA of individual cells. With this method, the present detection limit for, e.g. O6-ethyl-2'-deoxyguanosine (O6-EtdGuo) is of the order of 7 X 10(2) O6-EtdGuo molecules per diploid genome. Therefore, cells (e.g. from biopsy material) can now be monitored directly for the presence of specific carcinogen-DNA adducts, or with respect to their capacity to remove enzymatically such modified structures from DNA. In combination with transmission electron microscopy, MAB also permit the direct visualization of specific carcinogen-modified sites in DNA. Thus, O6-EtdGuo can be localized in double-stranded DNA molecules by the binding of a MAB specifically directed against this ethylation product.

Antibodies, Monoclonal↗
PubMed1983

Isolation and characterization of Drosophila melanogaster U2 small nuclear RNA genes.

We describe here the organization of DNA sequences complementary to Drosophila melanogaster U2 small nuclear (sn) RNA. From a genomic library we isolated two recombinants containing two genes each. Genomic reconstruction experiments and Southern analysis revealed that D. melanogaster possesses only four to five U2 snRNA genes or very closely related sequences. The nucleotide sequence of one of the clones analysed shows 77% homology with rat U2 snRNA. A stretch of 12 nucleotides that has been implicated in heterogeneous nuclear RNA splicing is conserved between rat and Drosophila. The genomic organization of these genes is very similar in different melanogaster strains but diverges highly in different Drosophila species.

Animals↗
PubMed1983

Fragmentation of 'nuclear matrix' on a mica target.

Chromatin depleted nuclei ('nuclear matrix') of Ehrlich ascites cells were characterized and fragmented by glycerol shot technique (particle fragmentation). The preparations reveal that 'nuclear matrix' is entirely composed of granules and fibres. Prominent size classes of granules are 10 to 20 nm and 25 to 40 nm, respectively. Most of the granules remain attached to fibres during the fragmentation process. The diameter of the fibres corresponds with double-stranded DNA visualized under identical conditions. The RNP-like nature of the particles is shown by their proteinase K/RNase sensitivity. Since the 'nuclear matrix' architecture becomes instable in high salt buffer after pretreatment with RNase which changes the RNP-particle-like material it must be inferred that the RNP/DNA interaction is a prerequisite for the high salt stability of the 'nuclear matrix' complex.

Animals↗
PubMed1982

Purified glucocorticoid receptor-hormone complex from rat liver cytosol binds specifically to cloned mouse mammary tumor virus long terminal repeats in vitro.

Purified glucocorticoid hormone-receptor complex (HRC) from rat liver binds to specific DNA sequences contained in cloned mouse mammary tumor virus (MMTV)DNA. The binding site of the hormone-receptor complex is located in the long terminal repeat (LTR) of MMTV DNA as shown by filter binding studies with labeled restriction fragments and by visualization of DNA-receptor complexes with the electron microscope. The DNAs from cloned MMTV lacking the LTR sequences were neither retained on nitrocellulose filters nor bound specifically to HRCs examined in the electron microscope. The HRC also failed to bind to restriction fragments from pBR322 and phage lambda. Specific binding of the HRC to LTR sequences is dependent upon occupancy of the receptor by a glucocorticoid. Previous work has demonstrated that the MMTV transcription is initiated within the LTR; additionally, MMTV transcription is known to be regulated by glucocorticoids. Our present results therefore support the hypothesis that HRC regulates hormone-induced transcription by binding to specific DNA sequences near the MMTV transcription start site.

Animals↗
PubMed1980

On the structure of crystalline ribulosebisphosphate carboxylase from Alcaligenes eutrophus.

Ribulosebisphosphate carboxylase from the hydrogen bacterium Alcaligenes eutrophus having a molecular weight of 534000 and consisting of eight large and eight small subunits has been crystallized by microdialysis using inorganic as well as organic precipitating agents. Crystals have tetragonal space group P42212, a = b = 11.27 nm, c = 20.14 nm, and contain one quarter molecule per asymmetric unit. X-rays are diffracted to 0.35-nm resolution on still photographs. Light optical diffractions of electron micrographs of thin sectioned crystals displayed patterns which could be interpreted on the basis of the unit cell determined by X-rays. Packing considerations are in accord with our earlier proposal regarding the subunit arrangement of this enzyme which differs from that reported for tobacco ribulosebisphosphate carboxylase.

Alcaligenes↗
PubMed1979

Structure of the 50S ribosomal subunit from Escherichia coli. Investigation of the intact subunit and core particles by electron microscopy and analogue image processing.

Structures of 50S ribosomal subunits, CsCl and ethidium bromide core particles from these subunits have been investigated by electron microscopy and image processing by FAIRS. This method revealed structural details which are obscured in individual images, and enabled to distinguish six crown forms, different in their side protuberances, and two kidney forms. Crown forms were imaged as symmetrical or asymmetrical forms. The latter type was far more frequent in untreated populations than the first. The depletion of proteins by both agents caused stepwise degradation of the side protuberances in the crown forms thereby transforming asymmetrical to symmetrical forms. It is concluded from these findings that asymmetrical and symmetrical forms in untreated populations represent also structurally different particles. From the higher complexity in terms of component composition and structure it is concluded that the asymmetrical crown forms are more likely to represent the native structure of isolated 50S subunits than the symmetrical forms. Existing models for this subunit are discussed in terms of this finding.

Escherichia coli↗
PubMed1978

Structure of LiCl core particles of 50 S ribosomal subunits from Escherichia coli by electron microscopy.

The structure of 50 S E. coli ribosomal subunits was studied by electron microscopy as these particles were gradually depleted of proteins by incubation with 0.5 to 6.0 m LiCl. Changes observed in the structure of the depleted subunits were correlated with the location of the deleted ribosomal proteins on the control 50 S particle. These changes were particularly striking in the "crown" region, the site of a considerable number of the proteins necessary for the biological activity of the 50 S subunit. Protein L 16, the first to be removed by the LiCl treatment, was found to be essential for the structural integrity of the large subunit through interactions with ribosomal proteins residing in the left-hand side crest and the interface. Based on electron microscopic evidence, a scheme was proposed for the structural changes accompanying the stepwise unfolding of the 50 S E. coli subunit by LiCl.

Bacterial Proteins↗