Biomedical subjects

E Spiess

Publications and source records attributed to E Spiess.

At least 37 records · Page 2Linked to original sources

PubMed1996

Differential secretion of cathepsins B and L from normal and tumor human lung cells stimulated by 12(S)-hydroxy-eicosatetraenoic acid.

Cathepsins B and L play roles in intracellular and extracellular proteolysis in normal and malignant processes. A directed extracellular proteolysis by regulated secretion could facilitate the process of invasion. We have therefore investigated the effect of the physiological signal mediator 12(S)-hydroxy-eicosatetraenoic acid on the release of cathepsins B and L in normal and malignant human lung cells. Quantitative determinations of cathepsin activities were done by flow cytometry and spectrofluorometry using synthetic dipeptidyl substrates coupled to fluorogens. Most interestingly, a difference in the secretion of cathepsins B and L was found: only release of active cathepsin B was detected. The effect was specific for 12(S)-hydroxy-eicosatetraenoic acid, 12(R)-hydroxy-eicosatetraenoic acid, and 5(S)-hydroxy-eicosatetraenoic acid were ineffective. The response was immediate but a substantial amount of nonreleasable activity remained cell bound. Alveolar macrophages, Wi-38 fibroblasts, and tumor cells derived from large cell carcinomas and adenocarcinomas were sensitive to 12(S)-hydroxy-eicosatetraenoic acid, but cells from undifferentiated squamous cell carcinomas were not. Sensitivity did not parallel malignancy but more likely the degree of differentiation of cells. The investigated tumor cell lines showed no detectable endogenous 12-lipoxy-genase activity to synthesize 12(S)-hydroxy-eicosatetraenoic acid from arachidonate; therefore, we assume a paracrine mechanism for 12(S)-hydroxy-eicosatetraenoic acid action. Protein kinase C alpha, a key enzyme involved in 12(S)-hydroxy-eicosatetraenoic acid-elicited responses, was expressed in all sensitive tumor cells, but insignificantly in a sensitive normal cell line and an insensitive tumor cell line. From our experiments we propose two separate intracellular pools of active cathepsin B: an unreleasable, lysosomal fraction and a fraction available for regulated secretion. Different processing and sorting mechanisms may be responsible for the generation of these cathepsin B-fractions in these pools.

12-Hydroxy-5,8,10,14-eicosatetraenoic Acid↗

PubMed1996

Three-dimensional analysis of the substrate-dependent invasive behavior of a human lung tumor cell line with a confocal laser scanning microscope.

Matrigel and collagen G gels were used as models for basement membrane and interstitial space-collagen, respectively, to study the invasive behavior of cells of the human lung tumor cell line EPLC 32M1, which was derived from a squamous cell carcinoma. For three dimensional analysis of the invasive process, cells were seeded onto the gels in a slide chamber and observed with a confocal laser scanning microscope. Optical sectioning in the xy and xz directions and image reconstruction with computer programs allowed us readily to obtain a three-dimensional overview of the invasive process in situ. Both types of gel showed a smooth surface. Matrigel had a granular structure whereas collagen G revealed a fiber-like morphology. The tumor cells showed a matrix-dependent behavior. On Matrigel, within 24 h of incubation, a network of cells appeared on the surface, which developed further within 72 h to interconnected multicellular cords also invading the gel. Tumor cells seeded on collagen G remained individual. They formed pseudopodia and achieved tight contact with the matrix, eventually also invading the gels in a time-dependent manner. Therefore, the composition of the substrate crucially influences the invasion path.

Animals↗

PubMed1996

Purification and characterization of chlorobenzene cis-dihydrodiol dehydrogenase from Xanthobacter flavus 14p1.

Chlorobenzene cis-dihydrodiol dehydrogenase was purified to homogeneity from Xanthobacter flavus 14p1, which used 1,4-dichlorobenzene as the sole source of carbon and energy. The enzyme converted a number of halogenated substrates with high specific activity. The pI of the native chlorobenzene cis-dihydrodiol dehydrogenase was 5.4, and the molecular mass was approximately 100 kDa, as determined by gel filtration. The enzyme was composed of four apparently identical subunits with a molecular mass of 26.5 kDa. The Michaelis constant for 3,6-dichlorobenzene cis-dihydrodiol (210 microM) was lower than for benzene cis-dihydrodiol (780 microM), while the specific activity with benzene cis-dihydrodiol (63 units/ mg) was higher than with 3,6-dichlorobenzene cis-dihydrodiol (32 units/mg). Chlorobenzene cis-dihydrodiol dehydrogenase accepted also NADP+ as cosubstrate; however, the activity was reduced to 14% of that with NAD+. The enzymic activity was inhibited by mercuric chloride and to a lesser extent by the metal-ion chelators 8-hydroxyquinoline and KCN.

Benzene Derivatives↗

PubMed1996

Tenascin-C contains distinct adhesive, anti-adhesive, and neurite outgrowth promoting sites for neurons.

The glia-derived extracellular matrix glycoprotein tenascin-C (TN-C) is transiently expressed in the developing CNS and may mediate neuron-glia interactions. Perturbation experiments with specific monoclonal antibodies suggested that TN-C functions for neural cells are encoded by distinct sites of the glycoprotein (Faissner, A., A. Scholze, and B. Götz. 1994. Tenascin glycoproteins in developing neural tissues--only decoration? Persp. Dev. Neurobiol. 2:53-66). To characterize these further, bacterially expressed recombinant domains were generated and used for functional studies. Several short-term-binding sites for mouse CNS neurons could be assigned to the fibronectin type III (FNIII) domains. Of these, the alternatively spliced insert TNfnA1,2,4,B,D supported initial attachment for both embryonic day 18 (E18) rat and postnatal day 6 (P6) mouse neurons. Only TNfn1-3 supported binding and growth of P6 mouse cerebellar neurons after 24 h, whereas attachment to the other domains proved reversible and resulted in cell detachment or aggregation. In choice assays on patterned substrates, repulsive properties could be attributed to the EGF-type repeats TNegf, and to TNfnA1,2,4. Finally, neurite outgrowth promoting properties for E18 rat hippocampal neurons and P0 mouse DRG explants could be assigned to TNfnB,D, TNfnD,6, and TNfn6. The epitope of mAb J1/tn2 which abolishes the neurite outgrowth inducing effect of intact TN-C could be allocated to TNfnD. These observations suggest that TN-C harbors distinct cell-binding, repulsive, and neurite outgrowth promoting sites for neurons. Furthermore, the properties of isoform-specific TN-C domains suggest functional significance of the alternative splicing of TN-C glycoproteins.

Amino Acid Sequence↗

PubMed1996

Structural analysis of the murine cell adhesion molecule L1 by electron microscopy and computer-assisted modelling.

In the present study we have analysed the morphology of two fragments with apparent molecular weights of 180 and 140 kDA (L1-180 and L1-140) derived from the extracellular region of the murine neural cell adhesion molecule L1. The fragment L1-180 consists of almost the entire extracellular part of the molecule, and is built up of six immunoglobulin-like and five fibronectin type III-like domains. Fragment L1-140 lacks one-half of the third, the fourth and the fifth fibronectin type III-like domains. By electron microscopic analysis of rotary-shadowed molecules, L1-140 and L1-180 revealed fibrillar structures 31-43 nm long and 7-12 nm wide with one pronounced globular terminal domain. As determined by complex formation with an L1 antibody, this terminal part of the molecule is formed by the fibronectin type III-like domains. The individual structures showed variation and complexity, and four distinct aspects were identified. These different forms probably represent two-dimensional projections of the same three-dimensional helical structure. Computer-assisted modelling of the L1 molecule, i.e. the protein backbone, showed no strong intramolecular interaction between the different fibronectin type III- or Ig-like domains, suggesting that the formation of the globular part of the molecule is probably achieved by protein-carbohydrate and/or carbohydrate-carbohydrates rather than protein-protein interactions. In addition, our model proposes that interactions occur within the interfaces between the different domains. The highly conserved amino acid residues in these regions point to the necessity of maintaining the orientation between the different domains.

Animals↗

PubMed1995

High salt- and SDS-stable DNA binding protein complexes with ATPase and protein kinase activity retained in chromatin-depleted nuclei.

Cell lysis in presence of SDS and proteinase K followed by salting-out of residual polypeptides by dehydration and precipitation with saturated sodium chloride solution [Miller, S.A., Dykes, D.D. and Polesky, H.F., Nucleic Acids Res., 16, 1215, 1988] efficiently resolves deproteinized DNA. However, this DNA is still associated with prominent polypeptides which remain stably attached to DNA during further treatments, e.g. during repeated salting-out steps, prolonged incubation of DNA in 1% SDS or 4 M urea at 56 degrees C and ethanol precipitation. The persistent polypeptides (62, 52 and 40 kDa) released from Ehrlich ascites cell DNA were further characterized. Microsequencing indicates that the DNA binding polypeptides are not yet characterized at the sequence level. Nuclease digestion of the DNA releases stable DNA-protein complexes with the shape of globular particles (12.8 +/- 0.8 nm) and their larger aggregates in which DNA remains protected from nuclease digestion. The isolated DNA-polypeptide complexes show ATPase (Km = 7.4 x 10(-4) M) and protein kinase activity. Antibodies reveal a parallel distribution of the complexes with chromatin, however, the complexes are retained in chromatin-depleted nuclei.

Adenosine Triphosphatases↗

PubMed1995

Degradation of 1,4-dichlorobenzene by Xanthobacter flavus 14p1.

Xanthobacter flavus 14p1 was isolated from sludge of the river Mulde by selective enrichment with 1,4-dichlorobenzene as the sole source of carbon and energy. The bacterium did not use other aromatic or chloroaromatic compounds as growth substrates. During growth on 1,4-dichlorobenzene, stoichiometric amounts of chloride ions were released. Degradation products of 1,4-dichlorobenzene were identified by gas chromatography-mass spectrometry analysis. 3,6-Dichloro-cis-1,2-dihydroxycyclohexa-3,5-diene and 3,6-dichlorocatechol were isolated from culture fluid. 2,5-Dichloromuconic acid and 2-chloromaleylacetic acid as well as the decarboxylation product 2-chloroacetoacrylic acid were identified after enzymatic conversion of 3,6-dichlorocatechol by cell extract. 1,4-Dichlorobenzene dioxygenase, dihydrodiol dehydrogenase, and catechol 1,2-dioxygenase activity were induced in cells grown on 1,4-dichlorobenzene. The results demonstrate that 1,4-dichlorobenzene degradation is initiated by dioxygenation and that ring opening proceeds via ortho cleavage.

Biodegradation, Environmental↗

PubMed1995

Assessment of cathepsin L activity by use of the inhibitor CA-074 compared to cathepsin B activity in human lung tumor tissue.

In a series of pairs of lung tumor tissue and non-tumor lung parenchyma from 50 patients, the activity of cathepsin L was measured with Z-Phe-Arg-AMC using the inhibitor CA-074 to delimitate from cathepsin B activity also present in the tissue extracts. Cathepsin B was assessed in the same samples with its specific substrate Z-Arg-Arg-AMC. It was found that in tumor tissue the median activities of cathepsin L and cathepsin B were increased 1.6-fold and 4.9-fold, respectively. The levels of activity of both enzymes did not correlate with TNM stages nor with cell differentiation of bronchial carcinomas. Cathepsin L activity was found to be insignificantly higher in adenocarcinoma compared to squamous cell carcinoma, while cathepsin B activity did not vary across the histologies. The activities of both enzymes were low in pulmonary carcinoids, which are known to be low-grade malignant neoplasms. The amount of cathepsin B activity exceeded by far that of cathepsin L activity as proven by measurement with Z-Phe-Arg-AMC in the presence of the inhibitor Z-Phe-Phe-CHN2:95-98% of cathepsin B activity vs 2-5% of cathepsin L activity were determined. By SDS-PAGE separation and immunoblot analysis, it could be demonstrated that significant amount of cathepsin L is complexed with the cysteine proteinase inhibitor kininogen. This explains the rather low cathepsin L activity values in the tissue extracts.

Adenocarcinoma↗

PubMed1995

Quantification of intracellular cathepsin activities in human lung tumor cell lines by flow cytometry.

The cysteine proteases cathepsin B and cathepsin L are very likely involved in invasive processes of normal and malignant cells, they become relevant for a number of diseases and are possibly prognostic markers for the outcome of human lung cancer. Therefore, we have determined activities of these related enzymes in cells and in cell extracts of human lung carcinoma cell lines of different cathepsin composition by flow cytometry and by spectrophotometry, respectively. To this end we applied the synthetic dipeptidyl substrates benzoxycarbonyl-arginyl-arginine- and benzoxycarbonyl-phenyl-arginine- coupled to 4-methoxy-beta-naphthylamide, aminomethyl-coumarine or rhodamine R110. The apparent enzymatic activities were differentially defined by protease inhibitors, particularly E-64 and CA-074. Independent of the dipeptidyl-composition more than 99 per cent of the apparent activity was due to cathepsin B when 4-methoxy-beta-naphthylamide or aminomethylcoumarine were the leaving groups. The 4-methoxy-beta-naphthylamide precipitate used for detection of cell associated activities revealed a wide spectrum of excitation to fluorescence thwarting the application of other possible fluorescent tags. Therefore, its application is restricted to uniparametric fluorescence investigations. Both dipeptidylgroups coupled to rhodamine R110 were promiscuous: only 25 to 30% of the apparent activity were due to cathepsin B; the predominant activity came from cathepsin L, irrespective whether intracellular or activities of cellular extracts were analyzed. However, rhodamine R110-coupled substrates open the way for multiparametric fluorescent analysis of cathepsins B and L containing cells if appropriate inhibitors for specification of the enzymatic activities are additionally applied. In very contrast to 4-methoxy-beta-naphthylamide, which causes irreparable damage to the cells, the rhodamine substrates permit studies with living cells and live cell sorting.

2-Naphthylamine↗

PubMed1994

Cathepsin B activity in human lung tumor cell lines: ultrastructural localization, pH sensitivity, and inhibitor status at the cellular level.

We investigated the appearance and activity of the cysteine proteinase cathepsin B and its physiological inhibitors, stefins A and B, at the cellular level in human tumor cell lines HS-24, derived from a primary lung tumor (squamous cell), and SB-3, derived from a metastasis (lung adenocarcinoma). In addition to cathepsin B, these tumor cells also expressed the immunologically and functionally related cathepsin L, but not cathepsin H. Stefin A was found in HS-24 but not in SB-3 cells; stefin B was found in both cell types. Using a specific fluorogenic cytochemical assay, the intracellular activity of the enzyme was localized and quantified. Thus, the cellular cathepsin B kinetics for the synthetic substrates Z-Arg-Arg-4M beta NA and Z-Val-Lys-Lys-Arg-4M beta NA, its pH dependence and inhibition by E64, stefins A and B, and cystatin C could be determined. From these measurements it appeared that the enzyme exhibited different cleavage rates for these substrates in the different cell types, showed considerable cleavage activity at neutral pH, which was stable under these conditions for extended time periods, and was highly sensitive to the inhibitors E64 and cystatin C but was considerably less sensitive to stefins, particularly stefin A. By conventional light microscopy, confocal laser scanning microscopy, and electron microscopy the enzymatic activity was localized in lysosomes, as expected, but also in the endoplasmic reticulum, nuclear membrane, and plasma membrane. The endoplasmic reticulum is a site at which only pre-mature enzyme forms exist, which are usually not active. The appearance of enzymatic activity at the plasma membrane confirms earlier biochemical and immunofluorescence microscopic investigations. The different sites of localization within the cells make it likely that different forms of the enzyme are expressed simultaneously, which follow alternate ways of processing and sorting. Taken together, the results support an involvement of the enzyme under extracellular conditions in degradative processes.

Amino Acid Sequence↗

PubMed1993

Functional topography of the myelin-associated glycoprotein. I. Mapping of domains by electron microscopy.

The functional topography of the myelin-associated glycoprotein (MAG) was investigated by electron microscopic analysis of rotary-shadowed molecules of a MAG fragment (MAG 90) comprising the five immunoglobulin-like domains of the extracellular part of the molecule. MAG 90 molecules appeared as rod-like structures (18.5 +/- 1.2 nm long and 4.0 +/- 0.8 nm wide) with a globular domain at one end. Antibodies directed against the amino- and carboxy-terminus of MAG 90 interacted with the non-globular terminal region, indicating that the molecule is bent in the globular region with the amino- and carboxy-terminal arms in close apposition to each other. An antibody which interferes with the binding of MAG to neurons interacted predominantly with the globular domain of MAG 90. The fibril-forming collagen types I, III and V bound mainly to the non-globular terminal region of MAG 90, whereas the majority of heparin molecules interacted with the globular region of the molecule. The L2/HNK-1 carbohydrate structure was localized at the non-globular region in the protein fragment comprising the fourth and fifth immunoglobulin-like domains.

Animals↗

PubMed1992

Interactions of the neural cell adhesion molecule and the myelin-associated glycoprotein with collagen type I: involvement in fibrillogenesis.

To gain insights into the functional role of the molecular association between neural adhesion molecules and extracellular matrix constituents, soluble forms of the myelin-associated glycoprotein (MAG) and the neural cell adhesion molecule (N-CAM), representing most of the extracellular domains of the molecules, were investigated in their ability to modify fibrillogenesis of collagen type I. MAG and N-CAM retarded the rate of fibril formation, as measured by changes in turbidity, and increased the diameter of the fibrils formed, but did not change the banding pattern when compared to collagen type I in the absence of adhesion molecules. Scatchard plot analysis of the binding of MAG and N-CAM to the fibril-forming collagen types I, II, III, and V suggest one binding site for N-CAM and two binding sites for MAG. Binding of MAG, but not of N-CAM, to collagen type I was decreased during fibril formation, probably due to a reduced accessibility of one binding site for MAG during fibrillogenesis. These results indicate that the neural adhesion molecules can influence the configuration of extracellular matrix constituents, thus, implicating them in the modulation of cell-substrate interactions.

Animals↗

PubMed1990

Karyotypic characterization of established cell lines derived from a squamous cell carcinoma and an adenocarcinoma of human lung cancers.

Two non-small cell carcinoma cell lines from the major histopathologic groups of human lung cancers have been karyotyped: HS-24 was established from a squamous cell carcinoma, and SB-3 was obtained from a metastasis of a poorly differentiated adenocarcinoma. Endoreduplication is characteristic for both cell lines. Subsequent loss of chromosomes led finally to hypotetraploid karyotypes with modal chromosome numbers of 66-68 and 70-72 for HS-24 and SB-3, respectively. The structural analysis was performed by G- and C-banding. Stable overrepresentation of chromosomes 7, 8, 12, and 16 was found. Both cell lines developed a characteristic set of disomic and stable markers. Chromosomes involved in markers were 1, 2, 5, 6, 10, 11, 16, and 17. Consistent numerical and structural normality for chromosomes 4, 18, and 21 was observed.

Adenocarcinoma↗

PubMed1990

Localization of cathepsin B in two human lung cancer cell lines.

We demonstrated the cysteine proteinase cathepsin B in two human lung tumor cell lines by cytochemical and immunocytochemical methods. The cell lines were derived from a squamous cell carcinoma of the lung (HS-24) and a metastasis to the adrenal gland from an adenocarcinoma of the lung (SB-3). For comparison and control, normal human lung fibroblasts cells (Wi-38) were also investigated. Intracellular cathepsin B activity was detected in all three cell lines. SB-3 and the normal fibroblast cells showed almost equal cathepsin B activity, which was considerably stronger than that in the HS-24 cells. Specific inhibitors for cathepsin B (E64, leupeptin, antipain) suppressed its activity completely. Stefin A, the physiological cathepsin B inhibitor, was less effective; this might depend on its limited penetrability into living cells. Localization of the cathepsin B was performed by conventional immunofluorescence microscopy and laser scanning microscopy. With specific anti-cathepsin B antibodies, the enzyme was localized in HS-24, SB-3, and Wi-38 fibroblast cells within perinuclear granules representing the lysosomal compartment. In the SB-3 cells, we additionally localized a minor fraction of the enzyme bound to the plasma membrane in a speckled distribution, accessible to the antibodies from the outside. This direct demonstration of cathepsin B distribution supports biochemical data about the dual localization of the enzyme in tumor cells. It also supports the possibility of a direct involvement of cathepsin B in the degradation of the extracellular matrix, and thus a contribution of the enzyme in invasion and metastasis.

Adenocarcinoma↗

PubMed1990

Detection of cathepsin B, plasminogen activators and plasminogen activator inhibitor in human non-small lung cancer cell lines.

Human non-small lung cancer cell lines HS-24 (established from a primary squamous cell carcinoma) and SB-3 (established from a metastasis of a primary adenocarcinoma of the lung into the adrenal gland) were analysed for the proteinases tissue-type plasminogen activator (tPA), urokinase-type plasminogen activator inhibitor (PAI-1). The proteinases were characterized by activity measurements, inhibition studies, enzyme-linked immunosorbent assay (ELISA), and Western blot analysis. Cell-associated proteinases were determined in cell lysates, secreted proteinases in cell conditioned culture media. Both cell lines were found to secrete uPA and PAI-1, whereas tPA could be detected only in HS-24 conditioned media. No cathepsin B activity could be detected in media of both cell lines. However, activation experiments and western blot analysis showed, that at least HS-24 secrete an inactive precursor. Cell lysates of HS-24 and SB-3 show PA activity, but on a low level. Cathepsin B activity was also found to be low in HS-24 lysates. However, SB-3 lysates show high cathepsin B activity. Further characterization of the proteinases by their sensitivity against several inhibitors suggests that they are similar to the corresponding proteinases of normal, nonmalignant cells.

Adenocarcinoma↗

PubMed1989

J1-160 and J1-180 are oligodendrocyte-secreted nonpermissive substrates for cell adhesion.

The glia-derived J1 extracellular matrix glycoproteins have been referred to as J1-160/J1-180 (the developmentally late appearing lower molecular weight group) and J1-200/J1-220 (the developmentally early appearing higher molecular group immunochemically related to tenascin). Members of the two groups show distinct cross-reactivities. To characterize the structural and functional differences between these J1 glycoproteins, two monoclonal antibodies were generated which recognize only the members of the lower molecular weight group. The two antibodies detect immunochemical similarities among the members of the lower molecular weight group, but do not react with J1/tenascin. J1-160 and J1-180 are specifically expressed by differentiated oligodendrocytes in culture and by myelin of the central nervous system and have not been found in the peripheral nervous system nor in any other organ of the adult mice tested. Electron microscopic examination of rotary-shadowed J1-160 and J1-180 reveals, respectively, dimeric and trimeric (tribrachion) kink-armed rodlike structures, which are linked by disulfide bridges. J1-160/J1-180 are nonpermissive substrates for the attachment and spreading of early postnatal small cerebellar neurons, astrocytes, and fibroblasts. In a mixture with laminin, J1-160/J1-180 are nonpermissive substrates for neurons, but not for astrocytes or fibroblasts. The repulsive effect toward neurons can be neutralized by one of the monoclonal antibodies, but not by the other. These observations are discussed in the context of cell interactions during regeneration in the mammalian nervous system.

Animals↗

PubMed1988

Structure of mouse DNA (cytosine-5-)-methyltransferase.

DNA (cytosine-5-)-methyltransferase was purified as a single polypeptide (190 kDa by SDS-PAGE) from mouse P815 mastocytoma cells. This enzyme transfers methyl groups to unmethylated as well as to hemimethylated DNA sites with a strong preference for the hemimethylated substrate. A structural analysis of the isolated enzyme by electron microscopical techniques was undertaken. On the basis of the results obtained, we propose a model for the enzyme structure. This model describes the enzyme as a hemi-elliptical globular structure with dimensions of 5.4-6.7 nm for the height h and 10.3-10.8 nm for the diameter d, respectively; this globular structure bears a small appendix at the flat side. A molecular mass of 235-250 kDa is calculated from the measured dimensions. Limited trypsin digestion of the enzyme led to a 160-kDa fragment which preserved the gross morphology of the original material. The possible structure function relationships are discussed.

Animals↗

PubMed1988

Distribution of O6-ethylguanine in DNA exposed to ethylnitrosourea in vitro as visualized by electron microscopy using a monoclonal antibody.

Application of a monoclonal antibody (Mab ER-6; Rajewsky et al., 1980) specific for O6-ethyl-2'-deoxyguanosine (O6-EtdGuo), in conjunction with a protein-free spreading procedure for double-stranded DNA molecules and transmission electron microscopy, permits the visualization of antibody molecules complexed to O6-EtdGuo residues formed in DNA upon reaction with the carcinogen N-ethyl-N-nitrosourea (EtNU) (Nehls et al., 1984). To obtain information on the distribution of O6-EtdGuo in native DNA exposed to EtNU in vitro, samples of purified rat brain DNA were briefly incubated with EtNU at concentrations differing by a factor of 8 (0.5 and 4 mg of EtNU/ml, respectively). As determined in DNA hydrolysates by competitive radioimmunoassay, the resulting DNA preparations contained O6-EtdGuo at O6-EtdGuo/2'-deoxyguanosine molar ratios of 15.1 X 10(-5) and 116 X 10(-5), respectively. Interspace distances between Mab-binding sites in both sets of ethylated DNA were determined by electron microscopy both in individual DNA fragments of different size, and in computer-generated, long-thread DNA constructs. Comparative statistical analyses by a newly developed MOLRANDO computer program show a non-random distribution pattern of Mab-binding O6-EtdGuo residues.

Animals↗