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M Sander

Publications and source records attributed to M Sander.

89 records · Page 5Linked to original sources
PubMed1986

Topoisomerase II cleavage in chromatin.

We have examined the effect of the anti-tumor drug VM-26 on purified Drosophila topoisomerase II, and used this drug to map (putative) topoisomerase II cleavage sites in chromatin. These studies indicate that VM-26 interferes with the strand breakage-rejoining catalytic cycle. VM-26 appears to stabilize the topoisomerase-II-cleavable complex and markedly enhances the formation of double-strand breaks in naked DNA. VM-26 also stimulates the formation of double-strand breaks in isolated Drosophila nuclei. Analysis of the parameters of the VM-26-stimulated cleavage reaction in nuclei strongly suggests that the double-strand scissions are generated by endogenous topoisomerase II. Finally, we have examined the distribution of (putative) cleavage sites for endogenous topoisomerase II in the chromatin of the 87A7 heat shock locus and the histone repeat unit. We have found that there are prominent VM-26-induced cleavage products from the 5' ends of the 87A7, the two heat shock protein 70 genes, and in the intergenic spacer separating these genes. Moreover, the pattern of VM-26-induced cleavage products is altered in nuclei prepared from heat-shocked cells. In the case of the histone repeat unit, only minor VM-26-induced cleavage products are observed in nuclei (in spite of the fact that experiments on naked DNA indicate that the histone repeat contains many major cleavage sites for purified topoisomerase II). These findings suggest that the nucleoprotein organization of different DNA segments may be important in determining whether specific sites are accessible to endogenous topoisomerase II in nuclei.

Adenosine Triphosphate↗
PubMed1986

Protein kinase C phosphorylates topoisomerase II: topoisomerase activation and its possible role in phorbol ester-induced differentiation of HL-60 cells.

DNA topoisomerase II from Drosophila was phosphorylated effectively by protein kinase C. With a Km of about 100 nM, the reaction was rapid, occurring at 4 degrees C as well as at 30 degrees C and requiring as little as 0.6 ng of the protein kinase per 170 ng of topoisomerase. About 0.85 mol of phosphate could be incorporated per mol of topoisomerase II, with phosphoserine as the only phospho amino acid produced. The reaction was dependent on Ca2+ and phosphatidylserine and was stimulated by phorbol esters. Calmodulin-dependent protein kinase II, but not cyclic AMP-dependent protein kinase, was also able to phosphorylate the topoisomerase. Phosphorylation of topoisomerase II by protein kinase C resulted in appreciable activation of the topoisomerase, suggesting that it may represent a possible target for the regulation of nuclear events by protein kinase C. This possibility is supported by the finding that the phorbol ester-induced differentiation of HL-60 cells was blocked by the topoisomerase II inhibitors novobiocin and 4'-(9-acridinylamino)methanesulfon-m-anisidide(m-AMSA), but not by the inactive analog o-AMSA.

Aminoacridines↗
PubMed1985

Drosophila topoisomerase II double-strand DNA cleavage: analysis of DNA sequence homology at the cleavage site.

In order to study the sequence specificity of double-strand DNA cleavage by Drosophila topoisomerase II, we have mapped and sequenced 16 strong and 47 weak cleavage sites in the recombinant plasmid p pi 25.1. Analysis of the nucleotide and dinucleotide frequencies in the region near the site of phosphodiester bond breakage revealed a nonrandom distribution. The nucleotide frequencies observed would occur by chance with a probability less than 0.05. The consensus sequence we derived is 5'GT.A/TAY decrease ATT.AT..G 3', where a dot means no preferred nucleotide, Y is for pyrimidine, and the arrow shows the point of bond cleavage. On average, strong sites match the consensus better than weak sites.

Animals↗
PubMed1985

Novel partitioning of DNA cleavage sites for Drosophila topoisomerase II.

We have examined the long-range distribution of double-stranded DNA cleavage sites for Drosophila melanogaster topoisomerase II. These studies reveal a novel partitioning of preferred topoisomerase II cleavage sites. In the eukaryotic DNAs examined, major cleavage sites were typically found in nontranscribed spacer segments and close to the 5' and 3' boundaries of genes. In contrast, there were few if any prominent cleavage sites within genes. In addition, most of the major topoisomerase II cleavage sites closely corresponded to naked DNA hypersensitive sites for the prokaryotic enzyme, micrococcal nuclease.

Animals↗
PubMed1984

A protein kinase activity tightly associated with Drosophila type II DNA topoisomerase.

A protein kinase activity has been identified that is tightly associated with the purified Drosophila type II DNA topoisomerase. The kinase and topoisomerase activities are not separated when the enzyme is subjected to analytical chromatography (phosphocellulose, single-strand DNA agarose, and Sephacryl S-300) and analytical glycerol gradient sedimentation. These two activities are also inactivated to the same extent by either heat or N-ethylmaleimide treatment. The evidence, however, does not rule out the possibility that the kinase activity resides in a polypeptide other than the topoisomerase polypeptide. The topoisomerase-associated protein kinase activity is not stimulated by Ca2+ or cyclic nucleotides. It shows a broad substrate range, including the DNA topoisomerase itself, casein, phosvitin, and histones. Phosphoamino acid analysis identified phosphoserine and phosphothreonine in polypeptides modified by the topoisomerase-associated protein kinase. No similar activity has been identified previously in Drosophila melanogaster.

Animals↗
PubMed1983

Double strand DNA cleavage by type II DNA topoisomerase from Drosophila melanogaster.

The purified type II DNA topoisomerase from the embryos of Drosophila melanogaster exists in its native form as a dimer of 170,000-dalton polypeptides. In addition to the 170,000-dalton polypeptides, 3 polypeptides with molecular weights of 151,000, 141,000, and 132,000 were resolved when the enzyme was analyzed by electrophoresis under denaturing conditions. All four polypeptides can participate in the topoisomerase cleavage reaction and form covalent complexes with the cleaved DNA. Furthermore, immunochemical and biochemical data showed that they are structurally related and, therefore, the smaller polypeptides are likely generated from the 170,000-dalton polypeptide by proteolysis. The double strand DNA cleavage reaction of Drosophila topoisomerase has different site specificity from the Escherichia coli DNA gyrase-effected reaction. However, they result in an identical DNA structure at the cleavage site, which is a staggered double strand break with 4-nucleotide long 5'-protruding ends. The 3'-ends at the site of cleavage by Drosophila topoisomerase II have free hydroxyl groups and can be extended by exactly 4 nucleotides with T4 DNA polymerase, while the 5'-ends are covalently linked to the topoisomerase molecules. This similarity in cleavage site structure for Drosophila topoisomerase II and E. coli DNA gyrase suggests that they share some fundamental features in their mechanism of action.

Animals↗
PubMed1981

Specificities of limulin and wheat-germ agglutinin towards some derivatives of GM3 gangliosides.

Lipid vesicles containing derivatives of GM3 ganglioside (II3-N-acetylneuraminosyllactosyl ceramide) were used to study the specificities of two lectins (limulin and wheat germ agglutinin) towards N-acetyl neuraminic acid and N-glycoloylneuraminic acid and some of their natural and chemically modified derivatives. The extent of the lectin binding to the gangliosides was related to the aggregation process of the lipid vesicles which was monitored as an absorbance increase. Limulin binds specifically to lipid vesicles containing N-glycoloyl derivatives of GM3. The hydroxyl group at C-4 and the carboxyl group of neuraminic acid have to be free for the binding to limulin. The side chain of neuraminic acid is not involved in the binding site of limulin. Wheat germ agglutinin binds to GM3 ganglioside only when the hydrophilic tail of the neuraminic group is cut off (C7 analogues). The acetamido group but not the carboxyl group is involved in the binding to wheat germ agglutinin. The wheat-germ-agglutinin-induced aggregation of vesicles containing derivatives of GM3-ganglioside is dependent on the pH, on the ionic strength and on the presence of Ca2+ ions. The dependence on ionic strength and Ca2+ is a consequence of the electrostatic repulsion of the vesicles. The wheat-germ-agglutinin-induced aggregation process of vesicles containing any suitable GM3-ganglioside derivative was reversed by the addition of N-acetylglucosamine showing that the N-acetylneuraminic acid derivatives bind to the N-acetylglucosamine binding site.

Calcium↗
PubMed1979

Quantitative serum lipoprotein electrophoresis in Nigerians.

In response to the growing demand for lipid profiles in Nigerian patients with suspected ischaemic heart disease, cerebro-vascular accident and hypertension, serum lipoprotein electrophoresis based on ozone-Schiff staining technique and densitometric scanning has been developed. Out of 93 serum samples 80 showed three distinct bands in the electrophoretic patterns; nine showed two distinct bands and four had unusual migration characteristics. Mean percentages of the three bands in the common pattern were 43.5, 22.8, and 33.7 for alpha pre-beta and beta lipoproteins respectively. The relative preponderance of alpha lipoproteins in Africans contrasts with the findings in Europeans. It supports the view that high serum alpha lipoproteins may be protective against ischaemic heart disease in contrast to the atherogenic role of beta lipoproteins.

Adolescent↗
PubMed1977

Serum lipids and lipoproteins in healthy Africans.

Using a simple precipitation technique we observed that the serum concentrations of low density lipoproteins in healthy Africans were less than half the serum concentrations in healthy Europeans. Serum concentrations of lipids and the lipoproteins varied according to the social class of Africans. Serum triglyceride concentrations did not rise with adult age but serum cholesterol concentrations did. Contrary to the findings in Europeans, the proportion of alpha lipoprotein fraction on serum electrophoresis in the Africans was relatively higher than that of beta lipoprotein fraction.

Adolescent↗
PubMed1977

Demonstration of 9-O-acetyl-N-acetylneuraminic acid in brain gangliosides from various vertebrates including man.

Ganglioside fractions were isolated from brains of man, cow, horse, pig, sheep, cat, rabbit, rat, chicken and codfish. The acylneuraminic acid residues, liberated from these gangliosides by treatment with dilute aqueous acid or neuraminidase, were analysed by the thin-layer chromatography and combined gas-liquid chromatography/mass spectrometry. Small amounts (up to 20%) of 9-O-acetyl-N-acetylneuraminic acid, and in bovine and porcine brain gangliosides also traces of N-glycoloylneuraminic acid, were found in addition to N-acetylneuraminic acid.

Animals↗
PubMed1976

[Dyspareunia].

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Dyspareunia↗
PubMed1976

Neuraminic acid-specific modification and tritium labelling of gangliosides.

1. A crude ganglioside mixture and pure GM1 and GD1a from bovine brain grey matter were prepared on a large scale. 2. The C7- and G8-analogues of NeuNAc were prepared from Collocalia mucoid and their structures established by gas-liquid chromatography and mass spectrometry. 3. Using model compounds in addition to various gangliosides, the conditions for the periodate oxidation and subsequent borohydride reduction of gangliosides were investigated with regard to the yield of C7- and C8-analogues of NeuNAc and the integrity of other monosaccharides in the oligosaccharide chain. These conditions were optimised to yield maximum C8-NeuNAc production and low C7-NeuNAc formation. Thus products were obtained which closely resemble the native gangliosides. 4. Using boro [3H] hydride, ganglioside derivatives with high specific radioactivity were prepared for the first time, containing either NeuNAc and labelled C8-NeuNAc or mainly labelled C7-NeuNAc depending on the prevailing conditions.

Animals↗
PubMedDate not supplied

Erythrocyte pyruvate kinase deficiency. The influence of physiologically important metabolites on the function of normal and defective enzymes.

The dependence of the erythrocyte pyruvate kinase (PK)-catalyzed reaction on the glycolytic intermediates glucose-6-phosphate (Gluc-6-P), 2,3-diphosphoglycerate (2,3-DPG) and the nucleotides ADP and ATP was studied in normal individuals and 14 patients with PK deficiency. The Gluc-6-P concentrations in the erythrocytes are markedly elevated (4- to 6-fold) in 9 patients with severe hemolytic anemia compared to those 5 exhibiting a mild clinical course (up to 2-fold increased). 2,3-DPG is elevated up to 2 times compared to the controls whereas the measured ADP and ATP only slightly deviate from the normal range. Control experiments showed that these elevations of Gluc-6-P and 2,3-DPG do not depend on the number of reticulocytes. In enzyme kinetic terms, Gluc-6-P shifts the Hill coefficient to smaller values, i.e. suppresses the positive cooperativity (sigmoidal reaction kinetics), found in normal and some of the mutant enzymes and shift the noncooperative enzymes of some patients to an enzyme exhibiting negative cooperativity. The negative cooperativity already present in the enzymes of some of the patients suffering from severe hemolytic anemia becomes more pronounced upon addition of Gluc-6-P. Apparently 2,3-DPG acts as an antagonist to Gluc-6-P in increasing the Hill coefficient, i.e. enhancing the positive cooperativity of the normal enzyme. It shifts the hyperbolic patients' enzymes to a sigmoidal reaction type and the enzymes of those patients with negative cooperativity to a hyperbolic type. ADP and ATP show a similar behavior as 2,3-DPG, but additionally inhibit the enzyme at higher concentrations. The influence of all four phosphates on the Michaelis constant varies depending on the type of cooperativity, in some cases increasing and in some cases decreasing K0.5 PEP. With 7 of the patients, all of them with severe clinical course, a genetic analysis of their R-type PK gene was performed and genetic defects have been identified in the coding sequence. The found changes in the amino acid sequence and their corresponding location in the tertiary structure of the PK subunit can satisfactorily explain the alterations of the regulatory properties of the mutant enzymes thus allowing to establish a good correlation between altered structural and functional properties of the deficient enzyme and the severeness of the course of the disease.

2,3-Diphosphoglycerate↗