Cell kinetic disturbances induced by treatment of human diploid fibroblasts with 5-azacytidine indicate a major role for DNA methylation in the regulation of the chromosome cycle

Summary BrdU-Hoechst flow cytometry was used to investigate the effects of DNA hypomethylation, induced by treatment with 5-azacytidine (5AC), on cell proliferation. When human fibroblast-like cells derived from skin and amniotic fluid were exposed to 5AC during three successive cell cycles, their clone-forming ability was diminished after removal of the drug. Treated cells were rendered quiescent by culture with low serum in the absence of the drug. Upon serum stimulation, they showed a diminished fraction of proliferating cells, which exhibited a prolonged transit through the S and G2 phase of the cell cycle, and a permanent arrest within the G2 compartment. This pattern of disturbed cell proliferation may in part explain the changes in replication banding pattern reported in the literature. Cytogenetic analysis of 5AC-treated cells revealed numerous endomitoses and tetraploid metaphases indicating a disturbed chromosome cycle in association with these cell kinetic perturbations.     

Photoreceptor-specific efficiencies of β-carotene,zeaxanthin and lutein for photopigment formation deduced from receptor mutant Drosophila melanogaster

Summary Drosophila rearing media had only -carotene, zeaxanthin or lutein as precursors for photopigment chromophores. Zeaxanthin and lutein are potentially optimum sources of the 3-hydroxylated retinoids of visual and accessory photopigments. Mutants made the electroretinogram in white (w) eyes selective for compound eye photoreceptors R1–6, R7 and R8: R1–6 domiantes w's electroretinogram; R7/8 generates w;ora's (ora = outer rhabdomeres absent); R8 generates w sev;- ora's (sev = sevenless). Microspectrophotometry revealed R1-6's visual pigment. In w, all 3 carotenoids yielded monotonic dose-responses for sensitivity (Fig. 4) or visual pigment (Fig. 7). An ultraviolet sensitivity peak from R1-6's sensitizing pigment was present at high but not low doses (Fig. 1). In w;ora, all 3 carotenoids gave similar spectra dominated by R7's high ultraviolet sensitivity (Fig. 2). For w sev;ora, all spectra were the shape expected for R8, peaking around 510 nm (Fig. 3). The sensitivity dose-response was at its ceiling except for low doses in w;ora (Fig. 5) and zero supplementation in w sev;ora (Fig. 6). Hence, without R1-6, most of our dose range mediated maximal visual pigment formation. In Drosophila, -carotene, zeaxanthin and lutein mediate the formation of all major photopigments in R1-6, R7 and R8.Abbreviations ERG electroretinogram - MSP microspectrophotometry - HPLC high pressure liquid chromatography - n.a. numerical aperture - w, sev, ora Drosophila mutants - y, p, r marg types of R7 and R8     

A laser-T-jump study of the adsorption of dipolar molecules to planar lipid membranes

The adsorption of the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) as well as of other dipolar molecules to the interface of artificial lipid membranes gives rise to a change of the dipole potential between the membrane interior and water. As a consequence of the adsorption of the neutral species, the conductance of planar membranes, observed in the presence of the macrocyclic ion carriers nonactin or valinomycin, may change by many orders of magnitude. Using this effect in combination with a laser-T-jump technique, the kinetics of the adsorption process were measured and were interpreted on the basis of a Langmuir-isotherm. A partition coefficient (at small concentrations) of _HA =4.7·10^–4 cm, a rate constant of desorption k _HA100 s^-1 and a maximum surface density N _D=7.7·10¹³/cm² were found. The concentration at half saturation is K _HA=2.7·10^-4 M.Using these values the membrane conductance induced by the ion carrier nonactin and the shape of the current-voltage relationship as a function of the ligand concentration in water was analyzed. A maxiumum dipole potential of V _D ^max =-239 m V and a contribution of b=3.1·10^-15V cm² per single adsorbed 2,4-D molecule was found. 74% of the dipole potential acts on the inner membrane barrier separating the two interfacial adsorption planes of nonactin. The remainder (26%) favours interfacial complex formation between nonactin and K⁺ from the aqueous phase. The data hold for membranes formed from dioleoyllecithin in n-decane.     

A putative replicative form of the abutilon mosaic virus (gemini group) in a chromatin-like structure

Summary The putative replicative form of the abutilon mosaic virus (AbMV), a geminivirus, was purified from infected plants. It was shown to consist of a bipartite genome of 2660 and 2640 bp. This double-stranded DNA has a closed or relaxed circular conformation and part of it is packed in nucleoprotein complexes with a chromatin-like structure. Similarities between the geminiviruses and the animal simian virus 40 are discussed against this back-ground.Cloning was performed under L2/B1 conditions according to the licence of the ZKBS 1526/1This article is based on a doctoral study by A.A. and T.F. in the Faculty of Biology, University of Hamburg     

Simulated nitrogen dynamics and nitrate leaching in a perennial grass ley

A soil nitrogen model was used for a 4-year simulation of nitrogen dynamics and nitrate leaching, both during grass ley growth and after ploughing a grass ley. Model results were compared with field measurements of soil mineral-N status and leaching. A soil water and heat model provided daily values for abiotic conditions, which were used as driving variables in the nitrogen simulation. Simulated values for mineral-N levels in the soil agreed well with field data for the first 3 years of the simulation. During the final year the model predicted considerably higher levels of soil mineral-N content compared with measurements. To reach the mineral-N level measured at the time of ploughing the ley, the simulated N-uptake by plants had to be increased by 8 g N m⁻². Simulations of nitrate leaching suggested that estimates of leaching based on measurements in tile-drained plots can be considerably underestimated. Accurate quantification of leaching in tile-drained plots often requires additional information on water-flow paths. A substantial increase in simulated and measured values for the mineral-N content of the soil occurred after ploughing the ley. In the simulation, most of the increase was due to a high crop residue input and the absence of a growing crop after ploughing. Litter accumulations in the soil during the 4-year period contributed little to the increase in soil mineral-N.     

Control of inspiratory duration in premature infants

We used single-breath mechanical loads and airway occlusions in premature infants to determine whether maturation influences the reflex control of inspiratory duration. We measured flow, volume, airway pressure, and surface diaphragmatic electromyogram (EMG) in 10 healthy preterm infants [33 +/- 1 (SD) wk gestation], 2-7 days of age. Three resistive and two elastic loads and occlusions were applied to the inspiratory outlet of a two-way respiratory valve. Application of all loads resulted in inspired volumes significantly decreased from control (P less than 0.001), and these decreases were progressive with increasing loads. Inspiratory duration (TI) was prolonged from control by all loads and occlusions when measured from the diaphragmatic EMG (neural TI) and by all but the smaller elastic load when measured from the flow tracing (mechanical TI). Similar decreases in inspired volume at the end of neural TI produced by application of both elastic and resistive loads resulted in comparable prolongation of neural TI. In contrast, for comparable volume decrements, resistive loading prolonged mechanical TI more than elastic loading (P less than 0.001). Mechanical and neural TI values of the breath after the loaded breath were unchanged from control values. Comparison of the neural volume-timing relationship in premature infants with our data in full-term infants suggests that the strength of the timing response to similar relative decrements in inspired volume is comparable. We conclude that reflex control of neural TI in premature infants depends on the magnitude of inspired volume and is independent of the volume trajectory.(ABSTRACT TRUNCATED AT 250 WORDS)     

Screening rabbit colonies for antibodies to Pasteurella multocida by an ELISA.

Rabbit serum samples from eleven different research facilities were evaluated for the presence of immunoglobulin G against Pasteurella multocida by using an enzyme-linked immunosorbent assay (ELISA). Each facility which submitted serum samples also provided a brief history of each rabbit colony tested. Rabbits from colonies reported to have endemic P. multocida or of undetermined status had 83 (58.9%) of 141 rabbits that were positive. Colonies reported to be free from P. multocida had 110 (92.4%) of 119 rabbits that were negative by ELISA. The ELISA test described here showed a high degree of agreement (92-94%) with two other P. multocida ELISAs at different diagnostic facilities. This study confirms that an ELISA testing for serum antibodies against the P. multocida is a reliable diagnostic tool to screen colonies for P. multocida.     

The mechanism of extrachromosomal homologous DNA recombination in plant cells

Summary By cotransfecting plasmids carrying particular mutations in the -glucuronidase (GUS) gene into Nicotiana plumbaginifolia protoplasts and by monitoring the recombination rates using a recently developed transient assay, we were able to obtain insights into the mechanism of extrachromosomal recombination operating in plant cells. An exchange of flanking markers takes place in over 90% of the recombination events. In most of the remaining cases two consecutive, independent single crossover events occur. These events involve the same DNA substrate and lead to two successive exchanges of flanking markers, thus mimicking a presumed double crossover intermediate. A comparison of the outcome of our experiments with the predictions of two recombination models originally proposed for mammalian cells indicates that extrachromosomal recombination in plant cells is best described by the single strand annealing model. According to this model all recombination events result in an exchange of flanking markers. Our results rule out the double strand break repair model which predicts that flanking markers are exchanged in only half of all events.     

Simulation of field scale denitrification losses from soils under grass ley and barley

Denitrification losses from soils under barley and grass ley crops were simulated. The model, which includes the major processes determining inputs, transformations and outputs of nitrogen in arable soils, represents a scale compatible with information generally available in agricultural field research. The denitrification part of the model includes a field potential denitrification rate and functions for the effect of soil aeration status, soil temperature and soil nitrate content. Easily metabolizable organic matter is assumed not to limit denitrification. Simulated values were compared with denitrification measurements made during two growing seasons in the barley and grass ley treatments of a field experiment in central Sweden.Calibration revealed that the optimal parameter values describing the effect of soil aeration on denitrification rates were similar for both treatments. The response function derived agreed well with two data sets found in the literature. The potential denitrification rate constant, derived in the simulations, was higher for grass ley than for barley, which was consistent with the differences in overall rates of carbon and nitrogen turnover found between treatments.The simulated mean denitrification rates for the two seasons were within 20% of the mean of the measured values. However, simulated denitrification showed less temporal variability and a less skewed frequency distribution than measured denitrification. Some of the measured denitrification events not explained by the model could have been due to the stimulating effects of soil drying/wetting and freezing/thawing on microbial activity.     

Streptomycetes possess peptidyl-prolyl cis-trans isomerases that strongly resemble cyclophilins from eukaryotic organisms

A functionally active 17.5 kDa peptidyl-prolyl cis-trans isomerase was purified to homogeneity from Streptomyces chrysomallus, a Gram-positive filamentous bacterium. Characterization of the enzyme revealed inhibition and binding characteristics, against the immunsuppressive drug cyclosporin A, which were similar to cyclophilins from eukaryotes such as mammals, plants, fungi and yeasts, but different from those of cyclophilins from enterobacteria such as Escherichia coli. The amino acid sequence of the S. chrysomallus cyclophilin, as deduced from the gene sequence, revealed a striking degree of amino acid sequence identity with the corresponding 17 kDa proteins of humans (66%), Neurospora (70%) and yeast (69%). Comparison with cyclophilin sequences from the Gram-negative enterobacteria revealed much less homology (25% identity with E. coli b, 23% identity with E. coli a). Cyclophilin was detected in each of the four other Streptomyces species tested. The cyclophilins from the various streptomycetes differed in size, varying between 17 and 20.5 kDa. The cyclophilins were abundant in the Streptomyces cells, and present throughout growth.