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E Delain

Publications and source records attributed to E Delain.

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

Covalent and non-covalent interaction of chymotrypsin with alpha 2-macroglobulin.

The pattern of covalent crosslinking between human alpha 2-macroglobulin (alpha 2M) and chymotrypsin has been investigated by chromatography and polyacrylamide gel electrophoresis in denaturing medium. Reaction with a single mol of chymotrypsin per mol alpha 2M results in the formation of a 95% covalent 1:1 chymotrypsin-alpha 2M complex and in the proteolytic cleavage of both 180 kDa monomers in one alpha 2M subunit. Proteolytic cleavage in the other alpha 2M subunit requires the presence of a second mol of chymotrypsin; part (20%) of the protease in the 2:1 chymotrypsin-alpha 2M complex thus formed appears to be non-covalently bound to the alpha 2M chains. Covalent binding is abolished when the reaction of alpha 2M with the protease is carried out in the presence of hydroxylamine. A single mol of the protease is then able to cleave all four 180 kDa monomers in alpha 2M.

Binding Sites↗
PubMed1987

The structure of tissue on cell culture-extracted thyroglobulin is independent of its iodine content.

The major protein synthesized in vitro by the ovine thyroid cell line OVNIS 6H is the prothyroid hormone thyroglobulin. Purified from serum-free cell culture media using sucrose gradient centrifugation, the thyroglobulin dimer was analysed for iodine content and observed by electron microscopy. In their usual medium, the OVNIS 6H cells produce a very poorly iodinated thyroglobulin containing 0.05 I atom per molecule. When cultured with methimazole or propylthiouracil, two inhibitors of iodide organification, less than 0.007 I atom/molecules was found. These molecules purified from cell cultures were compared to those purified from ovine thyroid tissue containing 26 I atoms/mol. Despite large differences in iodine content, the three preparations all consist of 19 S thyroglobulin dimers with the classical ovoidal shape. The variability in size measurements remains in a 2% range for all thyroglobulin types. Consequently, no real significant variation can be found between the highly iodinated thyroglobulin isolated from tissue, and the poorly or non-iodinated thyroglobulins isolated from cells cultured with or without methimazole or propylthiouracil.

Animals↗
PubMed1986

Ultrastructural and anti-proteinase activity modifications of human alpha 2-macroglobulin induced by zinc and other divalent cations.

Native tetrameric alpha 2-macroglobulin molecules (alpha 2M) can be converted into a population of dimers by incubation with various divalent cations such as Zn, Cd, Mg, Cu, Ni, Co. This dissociation is completed within 30 min at 37 degrees C. These dimers have a characteristic shape and a size of about 16 X 8 nm, and appear to be the half of the native alpha 2M molecule which has a clear tetrameric structure as seen in the electron microscope. At room temperature or below, dimers obtained with 5 to 100 mM Zn++ can reassociate in long linear polymers which display a regular chain-like arrangement and a helical periodicity. The structural characteristics of this polymer are described. The trypsin inhibitory capacity of Zn++-treated alpha 2M has been studied in an attempt to correlate its Zn++-induced conformational changes with its functional modifications.

Cations, Divalent↗
PubMed1985

Antibodies to Z DNA stabilized with polyarginine.

The left-handed form of poly(dG-m5dC).poly(dG-m5dC) induced by heating the copolymer in the presence of magnesium and stabilized with polyarginine can be used to raise antibodies in rabbits. These antibodies are able to recognize the Z conformation of both methylated and nonmethylated forms of the copolymers. In the same experimental conditions, hypermethylated B DNA is not recognized by these antibodies.

Animals↗
PubMed1985

The accessibility of 5-methylcytosine to specific antibodies in double-stranded DNA of Xanthomonas phage XP12.

Antibodies specifically directed to 5-methylcytidine were raised in rabbits and purified by affinity chromatography. The accessibility of 5-methyldeoxycytidine (m5dCyd) to such antibodies was studied with DNAs from various origins. The reaction was followed by measuring the retention of radiolabelled DNA by antibodies on nitrocellulose filters, by immunoprecipitation, by gel filtration and was visualized with the electron microscope. Antibodies did not bind to Escherichia coli B DNA, which is deficient in m5dCyd. Denatured and native DNA from calf thymus, which contains m5dCyd as a minor nucleoside, was weakly retained on the filters whereas DNA extracted from Xanthomonas oryzae XP12 bacteriophage, which is rich in m5dCyd, was well recognized even in the native form.

5-Methylcytosine↗
PubMed1985

Electron microscopy of the conformational changes of alpha 2-macroglobulin from human plasma.

High resolution electron microscopy reveals that fully active alpha 2-macroglobulin (alpha2M) from fresh human plasma presents a very characteristic tetrameric structure. This native conformation of the alpha2M molecule is described here for the first time, along with its various orientations in negatively stained preparations. Although the native form is sensitive to inactivation, glutaraldehyde fixation is not necessary for its observation except when ammonium salts are used. The tetrameric structure of alpha2M undergoes a drastic conformational change when the protein is treated either with trypsin, thrombin or methylamine, as evidenced by the appearance of the typical)+(structure already described in the literature. The various aspects of this second conformation correspond to different orientations of the molecules in the stain film, and depend upon the nature of the support.

Journal Article↗
PubMed1984

Different Z DNA forming sequences are revealed in phi X174 RFI by high resolution darkfield immuno-electron microscopy.

The specific interaction between left-handed Z DNA sequences in negatively supercoiled bacteriophage phi X174 replicative form I (RFI) DNA and anti-Z DNA immunoglobulin G (IgG) was investigated by high resolution darkfield immuno-electron microscopy. DNA-antibody complexes were formed and maintained under optimal binding conditions, purified by column chromatography, and visualized after uranyl acetate staining without using aldehyde fixation, shadowing, or second antibody. Bivalent anti-Z DNA IgGs bound to RFI molecules, thus forming intramolecular bridges. They could also oligomerize separate molecules by intermolecular linking of Z DNA sequences. At relatively low ionic strength and low temperature, high affinity anti-Z IgG was retained at certain loci even after restriction endonuclease cleavage of the DNA. In these cleaved molecules some superhelices could be visualized in the loops generated by the bivalent IgG. To our knowledge this is the first example of polypeptide stabilization of local superhelical strain in a cut molecule. Z DNA sequences in phi X174 RFI DNA were mapped. Alternating tracts of purines and pyrimidines starting at nucleotides 763, 1027, 1714, 2146, 2363, 3504, 4161, 4911 and 5345 occur within the nine different anti-Z IgG binding sites which were expressed with varying frequencies (53-3%) on the molecules. Usually, a limited number of sites (generally less than or equal to 2) exists on any one molecule. The formation of multiple Z sites (at the extracted superhelix density) in a given molecule is probably non-cooperative due to relaxation of torsional stress by the B----Z transition. Z sites occur in several different genes, including regions where transcription is attenuated and, in one case, in front of a promoter of transcription.

Antigen-Antibody Complex↗
PubMed1984

Electron microscopic measurement of chain flexibility of poly(dG-dC).poly(dG-dC) modified by cis-diamminedichloroplatinum(II).

The antitumor drug cis-diamminedichloroplatinum (II) (cis-Pt) forms bidentate adducts with guanine residues of poly(dG-dC).poly(dG-dC). The secondary structure of the polymer is altered. In this work, high resolution pictures of naked molecules, obtained by dark field electron microscopy reveal DNA chain distortions with radii as small as 30 A. The extent of distortion increases with the drug/nucleotide ratio (rb). These alterations of the secondary structure are responsible for the apparent shortening of the molecules. Measurements of the persistence lengths of the polymer as well as the end-to-end distances of elementary segments of various lengths, are obtained from digitized electron micrographs. The measurements are used to monitor and quantify the observed modifications of polymer structure upon cis-Pt binding at various rb or incubation times. Poly(dG-m5dC).poly(dG-m5dC) in the B and Z forms have different persistence lengths. In the B form, this polymer is more altered by cis-Pt than in the Z one.

Cisplatin↗
PubMed1983

Poly(ADP-ribose) polymerase auto-modification and interaction with DNA: electron microscopic visualization.

The interaction between purified calf thymus poly(ADP-ribose) polymerase and its activating co-purified DNA (sDNA) was investigated by electron microscopy. We have shown that the enzyme-DNA complex possesses a nucleosome-like structure. The enzyme-bound DNA (sDNA) was found to be enriched in single-stranded regions and branched structures, presumed to be replication forks. The auto-ribosylated polymerase as well as the branched poly(ADP-ribose) formed were visualized by dark field electron microscopy during the auto-ADP-ribosylation reaction and the possible mechanism of this phenomenon is discussed.

Animals↗
PubMed1983

Three dimensional association of double-stranded helices are produced in conditions for Z-DNA formation.

The Z form of alternating poly(dG-dC).poly(dG-dC) can be induced when the concentration of NaCl, MgCl2 or ethanol are increased. In order to obtain more information concerning this Z structure, the B----Z transition is analyzed on the same sample, both by UV spectrophotometry and electron microscopy. The procedures used in this work provide high resolution images with minimal alterations of the molecules. It is shown that at high values of cations or ethanol, the polymer makes complex associations of numerous molecules stuck together parallelly. By decreasing the salt or ethanol concentrations, a progressive decondensation of the molecules is obtained. At low concentrations of Mg++ (2.10(-2) M), alterations of the linear secondary structure of the molecules are observed, although the UV spectrum is of the B-type. In the presence of that low concentration of Mg++, natural DNAs (phi X174 and yeast mitochondrial DNA fragment inserted in pBR) exhibit structural modifications similar to those observed with the poly(dG-dC).poly(dG-dC). These structures mainly consist in four-stranded hairpins and loops built up by the sticking of two segments of DNA. The correlation between these intertwining of short DNA segments and the presence of potentially Z-forming sequences is discussed.

DNA↗
PubMed1983

Some electron microscopic aspects of poly(ADPR) polymerase-DNA interactions and of auto-poly(ADP-ribosyl)ation reaction.

Interaction of calf thymus poly(ADP-ribose(ADPR] polymerase with a copurified DNA fraction (sDNA) was investigated. Electron microscopic studies of sDNA which appeared to be a powerful poly(ADPR) polymerase activator have shown that the purified poly(ADPR) polymerase-DNA complexes possess a "nucleosome like structure", with DNA wrapping around the enzyme molecule. Examination of the DNA linked poly(ADPR) polymerase preparations revealed the presence of Y-structures in sDNA. The enrichment in the sDNA fraction of the Y shape DNA suggests the existence of replication fork structures in the poly(ADPR) polymerase linked DNA and or in the vicinity of the enzyme. With increasing auto-poly(ADP-ribosyl)ation the enzyme molecule becomes much denser, increases in size and detaches from the DNA. When poly(ADPR) formed was purified and examined by electron microscopy, branched polymers of different sizes were observed. The formation of these polymers may explain the size gained by poly ADP-ribosylated enzyme molecules. When the interaction of poly(ADPR) polymerase with the plasmid pBR 322 was tested, a slight contamination of our enzyme preparation with topoisomerase I was detected. The contaminant topoisomerase I activity, however, was completely abolished by ADP-ribosylation. Further experiments with purified calf thymus topoisomerase I confirmed that this enzyme loses its activity following ADP-ribosylation with poly(ADPR) polymerase. These results may suggest that ADP-ribosylation of topoisomerase I can be one of the regulatory mechanisms of its activity. Furthermore, these results confirm that a topoisomerase I contaminant does not interfere with the ADP-ribosylation experiments of purified poly(ADPR) polymerase preparation.

Adenosine Diphosphate Ribose↗
PubMed1981

Modification of lipophilic proteins in Friend erythroleukemia cells during their differentiation.

In mouse erythroleukemia cells (MELC) a lipophilic protein of apparent M.W. 9.5 kdaltons increases during differentiation. This increase is due either to an increase of biosynthesis or to a structural alteration impairing the capacity of the protein to form polymers of apparent high M.W. or favoring its extractability. The increases is related to differentiation and precedes hemoglobin synthesis by at least 1 day. It is not related to virus production because it occurs in cells (line F 4-1) which do not produce virus, but it does not occur in cells (TFA-II) in which dimethylsulfoxide (DMSO) causes an increase in virus production. As it occurs in cells treated with 4 different inducers, and as the increase is less marked when antagonists of the inducers are also present, it is unlikely that the increase of the 9.5 Kdalton protein is due to an effect of the inducers unrelated to differentiation.

Animals↗
PubMed1981

Improved visualization of single- and double-strained nucleic acids by STEM.

Annular dark field STEM images have been used to visualize nucleic acid molecules positively stained with uranyl acetate. The regular distribution of uranium clusters makes clearly visible the existence of segments of double and single strands on partially denatured RNA molecules. Unpaired regions, as short as about 8 nm, are detectable by this combination of a highly efficient imaging mode and a well adapted preparation technique.

DNA, Circular↗
PubMed1980

Electron microscopy of the reactions of anti-poly A. poly U and anti-poly I. poly C antibodies with synthetic polynucleotide complexes and natural nucleic acids.

The reactions between purified anti-poly A. poly U and-poly I. poly C. antibodies (IgG and IgM), and synthetic and natural polynucleotides were visualized at the molecular level. This was achieved by the use of fine tungsten bidirectional shadowing of molecules adsorbed onto thin carbon films, combined with dark field electron microscopic observation. A progression was observed from monogamous multivalency (binding of a single multifunctional antigen molecule with several combining sites of the same antibody molecule simultaneously) (Crothers and Metzger, 1972, Immunochemistry, 9, 341-357), to aggregation. Different types of figures were observed, among which loops formed by the coiling of the antigen around a single IgM molecule were very frequently seen. The tendency of IgG antibodies to bind cooperatively to certain antigens was also noted. In contrast, cross-links were seldom encountered. The cross-reactivity of different polynucleotides was also assessed by a quantitative analysis. The length of antigen associated to an antibody molecule (either IgG or IgM) was also measured.

Animals↗