Rudolf Beyer

Action of 1-β-d-Arabinofuranosylcytosine on mammalian tumor cells—1.

Abstract Synchronized mouse lymphoma cells were treated with ara-C- 3 H at the beginning of the S-phase with twice the ED 50 concentration. In DNA isolated from these cells ara-C could be traced. It could be shown that the radioactivity in nucleotide-and nucleoside fractions obtained after enzymic digestion of ara-C-DNA was due for more than 87% to ara-C- 3 H. The radioactivity of ara-C- 3 H can be almost quantitatively attributed to cytosine and arabinose. An incorporation of ara-C into RNA could not be detected.

9-β-d-Arabinofuranosyladenine as a tool to study herpes simplex virus DNA replication in vitro

Abstract 9-β- d -Arabinofuranosyladenine (araAdo) strongly suppresses herpes simplex virus (HSV) DNA synthesis in intact cell systems. After incubation with araAdo, two HSV-DNA fractions can be isolated by neutral isopycnic CsCl density gradient centrifugation, a light fraction with a buoyant density of 1.726 g/cm 3 , and a heavy fraction with a density of 1.738 g/cm 3 . After recentrifugation in neutral CsCl gradients, the light and heavy fractions are detected at a density of 1.729 and 1.741, respectively. Analysis of the two HSV-DNA fractions by hydroxylapatite chromatography and by digestion with nuclease S 1 revealed that the light fraction consists predominantly of native DNA and the heavy fraction of denatured DNA. AraAdo is incorporated into HSV-DNA and has been shown to be present in terminal positions at the 3′-hydroxyl position. The HSV-DNA pieces, containing incorporated araAdo, are of low molecular weight (less than 2.6 × 10 6 ) and are not assembled to higher molecular weight aggregates.

Age-correlated DNA damage in human muscle tissue

This investigation represents the largest study so far published on human DNA damage and aging. The subject of this investigation is damage, determined as DNA alterations which give rise to complete molecular breaks in the course of treatment of purified DNA solutions with single-strand-specific nucleases. The DNA is derived from milligram samples of human muscle of individuals mostly undergoing surgical treatment. Care has been taken to bring the muscle samples, once shut off from blood circulation to liquid nitrogen temperatures within few seconds. The DNA is prepared by a procedure keeping breaks by handling and by DNAase attack as low as possible, however pushing DNA purity, especially with respect to protein as high as possible. Highly purified DNA treated in this way has some sites which are susceptible to single-strand (ss) specific DNAase splitting (ss-events). Three different deoxyribonucleases have been used: Nuclease S1, Nuclease BAL31 and Pea Endo-Nuclease. They give very similar results, i.e. splitting of the DNA so as to yield DNA pieces of given distribution. The lengths of these double-strand (ds) pieces have been determined from their electron microscopical pictures, either by following the image contours with a magnetostrictive stylo of the projected photo on a pad, by following the contours with a mileage ruler, or by integrating the silver grains on the photo. The molecular weight averages of the ds DNA threads between two ss-events for each individual have been determined from 20 to 200 molecules. The 470 individuals contributing their data were from age groups from 1 to 91 years. The molecular weights show a considerable scatter with an average molecular weight of the DNA ds pieces between two ss-events of 43.93 MDa and a standard deviation of 17.99 MDa. Among the single-strand breaks (ssb) that split the DNA into such pieces is a fraction, the number of which increases in a highly significant fashion with the age of the donor. From this derives the fact that the average molecular weight of the DNA strand pieces between two ssb decreases with age. It is remarkable that the standard deviation of the molecular weights of such pieces increases with age significantly, too. On the basis of additional information mainly supplied by the DNA donor himself or by his parents the 470 members of the main group M where grouped according to their life-style, into: (1) abstinent people, essentially non-smokers and refraining from use of licit or illicit drugs, sub-group N.(ABSTRACT TRUNCATED AT 400 WORDS)

Species-specific aggregation factor in sponges. I. Characterization of the large circular proteid particle

Abstract We have isolated circular proteid particles (CPP) from the sponge Geodia cydonium , which carry the aggregation factor, and have freed them from concomitant impurities. The CPP have been visualized by electron microscopy as annular particles with a circular contour length of 3530 A. The circles are surrounded by 25 filaments 610 A in length. The CPP are characterized by a sedimentation coefficient ( S ° 20, w ) of 3000, by a buoyant density of 1.32 g/cm 3 and by a particle weight of about 2–5 · 10 9 daltons. They consist primarily of protein. It is suggested that the fibrous complex found electron microscopically is only the core structure of a more complex particle.

Arabinosyl nucleosides. XII. Influence of arabinofuranosylthymine on growth of L5178y cells

The antibiotic 1-beta-D-arabinofuranosylthymine (araThd) is a potent inhibitor of the growth of mouse lymphoma cells (L5178y). The ED50 concentration was found to be 9.8 muM. The cells die as a consequence of an unbalanced growth. The cytostatic activity of araThd can be abolished by coincubation with dThd and dUrd but not with Urd. At cytostatic concentrations araThd selectively blocks DNA synthesis; RNA- and protein synthesis are unaffected. Intracellularly araThd is rapidly phosphorylated to araTTP. This enzymic phosphorylation does not influence the synthesis of the naturally occuring, related triphosphate dTTP. AraTMP is incorporated into DNA during DNA synthesis; 1 mol of ara-TMP is incorporated/19,500 molecules of dTMP.

The effect of the cytostatic agent 9-α-d-arabinofuranosyladenine on the nucleic acid metabolism in L5178Y cells

Abstract The influence of 9-α- d -arabinofuranosyladenine (α-araAdo) on cell metabolism of mouse lymphoma cells (L5178y) has been studied on cellular as well as on subcecllular level. α-AraAdo strongly inhibits the cell proliferation of L5178y cells. Starting with 3 × 10 3 cells/ml and an incubation period of 72 hr, the drug reduces the cell proliferation to 50 per cent at a concentration of 12 μM. The cells die because of “unbalanced growth”. The cytostatic effect of α-araAdo can be abolished by coincubation with deoxyadenosine but not by adenosine. At cytostatic concentrations α-araAdo inhibits selectively the incorporation rate of thymidine into DNA but not the incorporation rates of precursors into RNA or protein. α-AraAdo is intracellularly phosphorylated to α-araATP. The compound has no effect on the extent of intracellular phosphorylation of exogenous administered adenosine or deoxyadenosine. α-AraAdo is incorporated into DNA but not into RNA; one molecule of α-araAdo is incorporated per 14,000 molecules of deoxyadenosine.

Arabinosyl nucleosides. XXXII. Mechanism of inhibition of L5178y mouse lymphoma cells by 9-β-D-arabinofuranosylguanine

The mechanism of the growth-inhibitory action of 9-beta-D-arabinofuranosylguanine (ara-G) on mouse lymphoma cells (L5178y) at the level of nucleic acid synthesis was studied. Ara-G inhibits the cell proliferation at an ED50 concentration of 12.3 microM. The cells die because of unbalanced growth. At cytostatic concentrations ara-G inhibits the incorporation rate of dThd into DNA to a much higher degree compared to those of the precursors into RNA or proteins. Ara-G has no effect on the induction of DNA polymerase alpha occurring at the beginning of the S-phase; this compound exerts its inhibitory activity during the S-phase. Ara-G is intracellularly phosphorylated to ara-GTP. It was demonstrated that ara-G is incorporated into DNA but not into RNA; one molecule of ara-G is incorporated per 7050 molecules of deoxyguanosine. The ara-GMP moieties, incorporated into DNA of L5178y cells are not removed during a culture period of 7.7 doubling steps; compared to the controls no reduction of the viability and growth rate was observed.

Highly protective alkalinization by ammonia vapor diffusion in viscosimetric DNA damage assessment

A method for the measurement of viscosities correlated to DNA alterations in alkaline homogenate suspensions is described. The alkaline pH shift to afford cell lysis, DNA unfolding, and denaturation is attained by gaseous ammonia diffusion, thus avoiding shear stress from mechanical mixing. At the same time a stabilizing density gradient is established. This solution is run through a plastic measuring tube that is wide enough to minimize the influence of uneven swelling of the lysing DNA-containing components. Flow times under a carefully controlled water head are registered, and their ratios to control solutions are evaluated. The relative viscosities show a strong and irreversible dependence on shear and on DNase treatment and therefore are considered as essentially DNA derived. The time dependence of the lysate viscosities with and without the DNA-damaging agent bleomycin is given and the dose:activity curves of this agent with sponge homogenates from two orders of Porifera are given with their 50% effective concentration values. The dose:activity curve of an extract from a polluted marine point source is demonstrated. The concentration changes in sponges exposed at differently polluted marine sites are shown. The idea of alkalinization through gaseous diffusion in conjunction with a simple measuring device has already proven a sensitive, reliable, and specific tool in the assessment of DNA damage produced under both laboratory and field conditions.

Arabinosyl nucleosides—XXXIII. Metabolism of 9-β-d-arabinofuranosyladenine in yeast cells

Abstract Growth of Saccharomyces cerevisiae is not affected by 9-β- d -arabinofuranosyladenine (β-araA) up to concentrations of 100 μM. Analytical studies revealed that β-ara-A is taken up by the yeast but is not intracellularly phosphorylated to the corresponding nucleotides; 27 per cent of the intracellular β-ara-A is deaminated to 9-β- d -arabinofuranosylhypoxanthine. However, incubation of the cells with ara-AMP results in a strong inhibition of cell proliferation with an ED50 concentration of 8.3 μM. The ara-AMP-caused inhibition of cell proliferation can be abolished by coincubation with deoxyadenosine. The latter observation together with results from incorporation studies indicates that ara-AMP affects cell growth via inhibition of DNA synthesis. In vitro experiments with partially purified adenosine kinase from yeast revealed that β-ara-A and eight further aranucleosides as well as three deaminase inhibitors (diazepin derivatives) are either not substrates or poor ones for this enzyme.