PubMed Health⌕ Search

Biomedical subjects

M Roberge

Publications and source records attributed to M Roberge.

49 records · Page 3Linked to original sources
PubMed1990

Nucleosome positioning as a critical determinant for the DNA cleavage sites of mammalian DNA topoisomerase II in reconstituted simian virus 40 chromatin.

We have assessed the ability of nucleosomes to influence the formation of mammalian topoisomerase II-DNA complexes by mapping the sites of cleavage induced by four unrelated topoisomerase II inhibitors in naked versus nucleosome-reconstituted SV40 DNA. DNA fragments were reconstituted with histone octamers from HeLa cells by the histone exchange method. Nucleosome positions were determined by comparing micrococcal nuclease cleavage patterns of nucleosome-reconstituted and naked DNA. Three types of DNA regions were defined: 1) regions with fixed nucleosome positioning; 2) regions lacking regular nucleosome phasing; and 3) a region around the replication origin (from position 5100 to 600) with no detectable nucleosomes. Topoisomerase II cleavage sites were suppressed in nucleosomes and persisted or were enhanced in linker DNA and in the nucleosome-free region around the replication origin. Incubation of reconstituted chromatin with topoisomerase II protected nucleosome-free regions from micrococcal nuclease cleavage without changing the overall micrococcal nuclease cleavage pattern. Thus, the present results indicate that topoisomerase II binds preferentially to nucleosome-free DNA and that the presence of nucleosomes at preferred DNA sequences influences drug-induced DNA breaks by topoisomerase II inhibitors.

Animals↗
PubMed1990

The topoisomerase II inhibitor VM-26 induces marked changes in histone H1 kinase activity, histones H1 and H3 phosphorylation, and chromosome condensation in G2 phase and mitotic BHK cells.

We have examined the effects of topoisomerase inhibitors on the phosphorylation of histones in chromatin during the G2 and the M phases of the cell cycle. Throughout the G2 phase of BHK cells, addition of the topoisomerase II inhibitor VM-26 prevented histone H1 phosphorylation, accompanied by the inhibition of intracellular histone H1 kinase activity. However, VM-26 had no inhibitory effect on the activity of the kinase in vitro, suggesting an indirect influence on histone H1 kinase activity. Entry into mitosis was also prevented, as monitored by the absence of nuclear lamina depolymerization, chromosome condensation, and histone H3 phosphorylation. In contrast, the topoisomerase I inhibitor, camptothecin, inhibited histone H1 phosphorylation and entry into mitosis only when applied at early G2. In cells that were arrested in mitosis, VM-26 induced dephosphorylation of histones H1 and H3, DNA breaks, and partial chromosome decondensation. These changes in chromatin parameters probably reverse the process of chromosome condensation, unfolding condensed regions to permit the repair of strand breaks in the DNA that were induced by VM-26. The involvement of growth-associated histone H1 kinase in these processes raises the possibility that the cell detects breaks in the DNA through their effects on the state of DNA supercoiling in constrained domains or loops. It would appear that histone H1 kinase and topoisomerase II work coordinately in both chromosome condensation and decondensation, and that this process participates in the VM-26-induced G2 arrest of the cell.

Animals↗
PubMed1988

RNA contacts the two large polymerase subunits and a 52-kDa polypeptide in nucleolar RNA polymerase I transcribing complexes.

The interactions between nascent RNA and RNA polymerase I-transcribing complexes were studied by photoaffinity labeling in HeLa nucleoli. Specific labeling of enzyme subunits Ia, Ib, and of a 52-kDa polypeptide was observed. The 52-kDa polypeptide was shown to be associated with the transcribing complex by immunoprecipitation with RNA polymerase I antibodies and by glycerol gradient centrifugation. Transcripts that were kept short by inclusion of a chain-terminating nucleotide or with short incubation times contacted mainly subunits Ia and Ib. Decreasing chain terminator concentration or increasing incubation time resulted in additional labeling of the 52-kDa polypeptide. These data indicate that the 52-kDa polypeptide contacts nascent RNA after it has left the Ia-Ib domain.

Cell Nucleolus↗
PubMed1988

Chromosomal loop/nuclear matrix organization of transcriptionally active and inactive RNA polymerases in HeLa nuclei.

The relative distribution of transcriptionally active and inactive RNA polymerases I and II between the nuclear matrix/scaffold and chromosomal loops of HeLa cells was determined. Total RNA polymerase was assessed by immunoblotting and transcribing RNA polymerase by a photoaffinity labeling technique in isolated nuclei. Nuclear matrix/scaffold was isolated by three methods using high-salt, intermediate-salt or low-salt extraction. The distribution of RNA polymerases I and II were very similar within each of the methods, but considerable differences in distributions were found between the different preparation methods. Either intermediate-salt or high-salt treatment of DNase I-digested nuclei showed significant association of RNA polymerases with the nuclear matrix. However, intermediate-salt followed by high-salt treatment released all transcribing and non-transcribing RNA polymerases. Nuclear scaffolds isolated with lithium diiodosalicylate (low-salt) contained very little of the RNA polymerases. This treatment, however, caused the dissociation of RNA polymerase II transcription complexes. These results show unambiguously that RNA polymerases, both in their active and inactive forms, are not nuclear matrix proteins. The data support models in which the transcriptional machinery moves around DNA loops during transcription.

Affinity Labels↗
PubMed1986

The head activator is released from regenerating Hydra bound to a carrier molecule.

Hydra forced to regenerate a head releases head activator and head inhibitor during the first hours after cutting to induce head-specific growth and differentiation processes. Analysis of the size distribution demonstrated that the head-activator peptide is co-released with (a) large molecular weight carrier molecule(s) to which it is non-covalently bound. The carrier-bound head activator is fully active on Hydra indicating that a carrier does not hinder the interaction with receptors. In contrast to this the head inhibitor is released in its naked, low molecular mass form. The association or non-association with a carrier molecule results in marked differences in biological properties. The head activator has a short range of action, but a long half-life, the head inhibitor has a global range of action, but a short half-life. These results provide a plausible explanation why two antagonistically acting substances, although they are released from the same site and simultaneously nevertheless can give rise to a well-defined temporal and spatial pattern of differentiation as occurs, for example, during head regeneration in Hydra.

Journal Article↗
PubMed1984

The hydra head activator in human blood circulation. Degradation of the synthetic peptide by plasma angiotensin-converting enzyme.

Using methanol extraction combined with HPLC and a new radioimmunoassay, the peptide head activator was detected in human plasma at a concentration of 20-100 fmol/ml. Synthetic head activator incubated with plasma was degraded with a half-life of 7 min. Analysis of sites of enzymatic cleavage and inhibition by captopril showed a major involvement of angiotensin-converting enzyme in this process. Endogenous head activator, on the other hand, was not appreciably degraded upon incubation of plasma in vitro. These findings raise the possibility that the endogenous peptide could bind to a protective carrier molecule and reach potential target tissues via the blood circulation.

Chromatography, High Pressure Liquid↗
PubMed1984

Cytological effects of ionophore-induced stimulation on the exocrine pancreas of the rat.

Rat-pancreas lobules were incubated with the ionophore A-23187 in the presence of Ca2+. After 90 min, some of the acini were partially or almost completely depleted of their zymogen granules while others had the appearance of resting acini. With few exceptions, the cells of a given acinus were degranulated to a comparable level. Slight dispersion of the zymogen granules was noticed in cells incubated in a Ca2+-free medium containing EGTA with or without A-23187. In the presence of Ca2+ the secretory response obtained with the ionophore was comparable to that observed with 10(-5)M urecholine. The results obtained provide cytological evidence that the secretory response is only partially determined at the membrane-receptor level and that other mechanisms intervene between cytosol Ca2+ increase and exocytosis.

Amylases↗
PubMed1983

The origin of the zymogen granule membrane of the pancreatic acinar cell as examined by ultrastructural cytochemistry of acid phosphatase, thiamine pyrophosphatase, and ATP-diphosphohydrolase activities.

Cytochemical distributions of acid phosphatase, thiamine pyrophosphatase, and ATP-diphosphohydrolase activities have been examined on thin sections of rat pancreas and on isolated zymogen-granule membranes. Acid phosphatase was found in the rigid lamellae separated from the Golgi stacked cisternae, in condensing vacuoles, and in the trans-saccules of Golgi apparatus; it was not detected in purified zymogen-granule membranes. Thiamine pyrophosphatase was detected in trans-saccules of the Golgi apparatus, in purified zymogen-granule membranes, and in the plasmalemma of the acinar cell. It was absent in condensing vacuoles. The ATP-diphosphohydrolase activity has a distribution similar to thiamine pyrophosphatase. These observations illustrate the similarity between the trans-saccules of the Golgi apparatus and the membrane of mature zymogen granules and the disparity between the latter membrane and the membrane of the condensing vacuole. They suggest that the condensing vacuole might not be the immediate precursor of the zymogen granule as commonly assumed. An alternative possibility would be that condensing vacuoles would fuse with the trans-saccule (transition) of the Golgi apparatus which in turn would form mature zymogen granules.

Acid Phosphatase↗
PubMed1982

Newly synthesized secretory proteins from pig pancreas are not released from a homogeneous granule compartment.

The pancreatic secretion of anesthetized pigs was collected by cannulation after pulse labeling with [3H]leucine. Collection at 5 min intervals started immediately post-pulse labeling up to 85 min. The volume, the protein content and the trichloroacetic acid-precipitable radioactivity of the juice were measured. The specific radioactivity of the secretory proteins was compared to that of a zymogen granule fraction isolated from the same animal. The latter was very much higher. Caerulein stimulation for 5 min at 80 min post-pulse caused a sharp drop in the specific activity of secretory proteins in the juice, to a level lower than that of the zymogen granule content. These data support the concept of more than one pool of secretory proteins in the pancreas and are incompatible with the concept that secretory proteins derive from an homogeneous granule compartment in a functionally homogeneous population of cells. To explain our results the hypothesis of a second intracellular route for the secretory proteins in proposed.

Animals↗
PubMed1982

Isolation of zymogen granules from rat pancreas and characterization of their membrane proteins.

A zymogen granule fraction has been isolated from rat pancreas, and its purity has been assessed by biochemical and morphological criteria. Specific activities of two marker enzymes, amylase and chymotrypsin, are increased by 4.6 and 5.4-fold, respectively, as compared to the homogenate. The purified fraction is devoid of detectable RNA, DNA and 5'-nucleotidase, glucose-6-phosphatase, and cytochrome c oxidase activities. Electron micrographs confirm the absence of mitochondria, lysosomes, and rough endoplasmic reticulum fragments. Zymogen granule membranes were isolated from this fraction on a sucrose gradient following lysis in alkaline buffer. Secretory contaminants were efficiently removed from the membranes as indicated by experiments in which labeled secretory proteins were added during the isolation procedure and secondly by measuring residual levels of amylase and chymotrypsin. Three enzyme activities were found in the membranes: thiamine pyrophosphatase, ATP-diphosphohydrolase, and low levels of acid phosphatase. Membrane proteins were solubilized by urea-Triton X-100 and separated in double-dimension (isoelectric focusing and sodium dodecyl sulfate-polyacrylamide gel electrophoresis). Isoelectric point and molecular weight of each protein band were determined.

Animals↗
PubMed1981

Cytological effects of urecholine stimulation on the rat pancreas.

Stimulation of the exocrine pancreas by the secretagogue urecholine caused degranulation of the acinar cells. Under in vivo conditions, this degranulation is not uniform throughout the tissue. Indeed some of the acini are almost completely depleted of their granules while others display the appearance of resting acini. A noticeable feature is that all the cells of the same acinus display a comparable degree of degranulation. Moreover, groups of neighbouring acini seem to respond simultaneously suggesting that the secretory stimulus is propagated from one acinus to the other. In vitro stimulation of dispersed acini also showed that some of the acini are more responsive than others indication that this phenomenon can not be attributed to accessibility of the secretagogue to its receptor. These observations lead us to accessibility of the secretagogue to its receptor. These observations lead us to the concept that the response of the pancreatic acinar cell is controlled at the level of the acinus.

Animals↗