Rudolf K. Zahn

Darstellung und längenmessungen des gesamten desoxyribonucleinsäure-inhaltes von T2-bakteriophagen

Abstract Electron micrographs of DNA macromolecules are prepared by spreading a suitable protein-salt solution with T2 phages to a mixed monolayer on a water-air interface. During the development of the film an osmotic shock occurs which confines the whole DNA content as a single thread near the phage ghost. The surface film is transferred to the support (carbonized formvar) by adsorption and removal of the water. The contrast arises by platinum deposition during the rotation of the specimens in a shadow-casting chamber. With a frequency of about 30%, unequivocal measurements of the DNA-thread system are possible. The first data of an average T2-DNA length of 49±4 μ are discussed in relation to the possible sources of error.

Benzo(a)pyrene monooxygenase induction in marine fish-molecular response to oil pollution

Induction of benzo (a) pyrene monooxygenase (BPMO) activity occurred in Blennius pavo, a species with a restricted territorial range, in response to exposure to a Diesel 2 oil. A response delay of 14 days was found at a concentration of 170 ppb and of 3 days when the water was saturated with Diesel 2 oil. When induced fish were transferred to clean water, elevated BPMO activity was maintained at a high level for at least a month. A benthic protochordate, Microcosmos sulcatus, showed no increase in BPMO activity when exposed to these concentrations even after 30 days of exposure. Field observations revealed a great variation in the BPMO activity from B. pavo caught at different sites. Fish from contaminated sites had significantly elevated levels of BPMO activity. Sardine schools caught at different sites had different, low levels of BPMO activities. However, specimens from the same school had closely similar levels of enzyme activity. An oil pollution incident (New Year 1977 oil spill in Northern Adriatic Sea) caused an increase in the BPMO activity in the livers of Blenniideae, reaching a peak on the 23rd day (representing an 8.5-fold increase in the background level), followed by a decrease in activity until a new background level, 3 times that of the original background level, was reached on the 45th day. This new background level is constant (through May-5 months after the incident). Measurement of BPMO activity in the livers of nonmigrant fish could serve as a useful biochemical parameter for monitoring and evaluation of acute or long-term oil pollution at a given site.

A microplate version of the SOS/ umu-test for rapid detection of genotoxins and genotoxic potentials of environmental samples

The umu-microtest is a miniaturized automated short-term test version proposed for screening of umuC-dependent mutagenic potentials of chemicals relevant to environmental pollution, river water and industrial waste water. The test is based on the SOS/umu-test and has been modified in order to allow extensive testing of environmental samples. Genetically engineered Salmonella typhimurium (TA1535/pSK1002) are incubated on a microplate rotor in a sloping position for 2 h with the test samples, followed by addition of fresh culture medium to reach a 10-fold dilution of the incubation medium. 2 h later, the activity of the beta-galactosidase, which reflects umuC induction, is determined colorimetrically. The incubation of the bacteria in the presence of the test compounds as well as the assessment of beta-galactosidase activity takes place in 96-well microplates, thus enabling simultaneous screening of large numbers of samples. Data of the genotoxic potentials are available within 8 h. Computer-controlled automation is possible by using a laboratory workstation.

Poly ADP-ribosylation of DNA-dependent RNA polymerase I from quail oviduct. Dependence on progesterone stimulation.

SummaryProgesterone causes in goblet cells of oviducts of estrogen hormone-stimulated immature quails selectively gene activation without affecting DNA synthesis. This biological model has been used to study the influence of poly ADP-ribosylation during the processes of DNA transcription.Administration of progesteronein vivo causes an increase of the activity of RNA polymerase I and II in isolated nuclei. This increase is accompanied by a marked decrease of the specific activity of poly (ADP-Rib) polymerase.Afterin vitro ADP-ribosylation of nuclear proteins the template capacity of chromatin for “exogenous” RNA synthesis (withE. coli DNA-dependent RNA polymerase) as well as for “endogenous” RNA synthesis with DNA-dependent RNA polymerase II is not affected, whereas the data presented seem to indicate that the capacity for RNA synthesis mediated by “endogenous” DNA-dependent RNA polymerase I might be inhibited after ADP-ribosylation.Evidence is presented to show that a considerable amount of poly (ADP-Rib), synthesized by poly (ADP-Rib) polymerase in isolated nuclei, is linked with RNA polymerase I. The rate of synthesis of poly (ADP-Rib) is dependent on the incubation temperature (optimum at 25°C) and it can be inhibited by the specific inhibitors of poly (ADP-Rib) polymerase nicotineamide, thymidine and formycin B. Poly (ADP-Rib) is probably associated with RNA polymerase I through a covalent linkage. ADP-ribosylated RNA polymerase I has been purified 550 fold with respect to the nuclear extract, corresponding to a 4,000 fold purification from the whole cell homogenate. The ratio between poly (ADP-Rib), formed during preincubation of nuclei with NAD, and RNA polymerase I remains almost constant during the purification procedures.The extent of ADP-ribosylation of RNA polymerase I decreases during gene expression. Thus we conclude that poly ADP-ribosylation of this enzyme is one of the regulatory mechanisms by which specificity of DNA transcription is achieved.

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.

Identification and properties of the cell membrane bound leucine aminopeptidase interacting with the potential immunostimulant and chemotherapeutic agent bestatin

Bestatin was found to be a competitive inhibitor (with respect to the Leu-NA substrate) not only of the isolated microsomal and cytosolic leucine aminopeptidases (Leu-APm and Leu-APc) but also of the aminopeptidases (APs) present in membrane preparations (from mouse liver) and on the cell surface of L5178Y cells. Kinetic parameters indicate that cellular AP is identical to Leu-APm. To rule out the possibility that AP-B is involved in the inhibition reactions, comparable studies with amastatin were performed. Electrophoretical studies revealed the solubilized cell membrane bound AP to co-migrate with Leu-APm in polyacrylamide gels. The activity of the separated membrane AP was inhibited by bestatin in situ. The cell membrane bound AP activity was found to be lowest in lymphocytes, higher in tumor cells and highest in bone marrow cells and macrophages. Using synchronized L5178Y cells, the AP activity changes during the cell division cycle; the lowest activity was determined during the G1-phase and 35% higher values were measured during the S/G2-phase. The fluctuation of the cell surface associated AP activity parallels with changes in the number of binding sites for bestatin.

DNA damage determination by the alkaline elution technique in the haemolymph of mussel Mytilus galloprovincialis treated with benzo[a]pyrene and 4-nitroquinoline-N-oxide

Abstract The ability of benzo[ a ]pyrene (BaP) and 4-nitroquinoline- N -oxide (NQO) to induce DNA breakage in haemolymph of mussel Mytilus galloprovincialis was studied using an alkaline elution assay. The elution of haemolymph DNA from control animals increased from pH 12.1–12.8 suggesting that DNA alkali-labile sites are already present in the haemolymph. The difference between control and treated ones is sufficient at pH 12.3 that additional alkali-labile sites and single-strand breaks caused by BaP and NQO can be measured. Elution of haemolymph DNA, from both BaP and NQO treated mussels, was found to increase in a dose-dependent manner after 1.5 h, followed by decrease in the elution rate depending on dose, after 48 h for BaP and 5 days for NQO. The results indicate that repair of DNA in mussel haemolymph is significant and dependent on the dose of chemical applied. DNA damage in BaP treated mussels could be explained by formation of peroxides and/or radicals during low BaP metabolism, and their effects on DNA.

Activation of DNA metabolism in T-cells by bestatin.

Abstract Bestatin, [(2S, 3R)-3- amino -2- hydroxy -4- phenylbutanoyl ]- l - leucine , is a microbial product which selectively influences the DNA metabolism of lymphoid tissues in vivo . The present studies, using CBA/J mice, revealed that bestatin increases the incorporation rate of dThd into DNA in spleen, thymus and bone marrow, but not in liver, kidney, spinal cord and lung. The stimulation was found to be dose-and timedependent, and it occurs only in T-cells, not in B-cells, from spleen and thymus. In addition, it is shown that bestatin causes a several-fold induction of DNA polymerase α only in T-cells from spleen and thymus, while the level in B-cells remains constant. Bone marrow cells respond to bestatin treatment with an increase of the DNA polymerase α activity and with an 8-fold induction of terminal deoxynucleotidyl transferase.

CONFIGURATION AND LONGITUDINAL DISTRIBUTION OF DNA MOLECULES IN SOLUTION

Abstract A diffusion method is described for visualization of DNA molecules which is equivalent to the spreading of a mixed DNA-protein solution. Macromolecules diffusing in a solution of about 5×10−8 g DNA per ml are adsorbed by a cytochrome c-film at the air-water interface. Subsequently they are transferred to electron microscopic supports. In the enlarged two-dimensional image the end-to-end distances and the length distribution are measured for different DNA preparations (from cod fish, trout, holothuria and T2 phage). Following recalculation in the three-dimensional state a relation is given for the mean square of the end-to-end distances, h 2 , and the length, L, as ( h 2 ) 1 2 = 1.3 × 10 −1 L(1 + √ 1 + 2.3 × 10 −3 L at ionic strengths from 0.09 to 0.35, according to a theory by Katchalsky and Lifson for diluted linear polyelectrolytes. The molecular shape is not a random coil or a rod but intermediate on account of electrostatic interaction. An application of the results on the theory of Kuhn, Kuhn and Buchner yields equations for the diffusion coefficient or other characteristics of DNA as a function of length or molecular weight. The length distribution of DNA, after disintegration by preparation and storage, usually has the form of a descending exponential function. Within the accuracy of the length distribution it is allowed to deduce random scissions along the native DNA molecule.

Two different aggregation principles in reaggregation process of dissociated sponge cells (Geodia cydonium)

Chemisch dissoziierte Zellen des KieselschwammesGeodia cydonium reaggregieren aufgrund zweier verschiedenr Reaggregationsprinzipien. Der Aggnegationsfaktor, auf den die Primäraggregation zurückgeht, ist membrangebunden und wird durch Proteasen nicht inaktiviert. Der sekundäre Aggregationsfaktor wurde 500fach angereichert. Das Molekulargewicht dieses Aggregationsfaktors beträgt etwa 20000 Daltons; er ist mit einem ringförmigen Makromolekül (2×109 Daltons) assoziiert.