Influence of Protein and Ribonucleic Acid Synthesis on the Replication of the Bacteriocinogenic Factor Clo DF13 in Escherichia coli Cells and Minicells

The influence of ribonucleic acid (RNA) and protein synthesis on the replication of the cloacinogenic factor Clo DF13 was studied in Escherichia coli cells and minicells. In chromosomeless minicells harboring the Clo DF13 factor, Clo DF13 deoxyribonucleic acid (DNA) synthesis is slightly stimulated after inhibition of protein synthesis by chloramphenicol or puromycin and continues for more than 8 h. When minicells were treated with rifampin, a specific inhibitor of DNA-dependent RNA polymerase, Clo DF13 RNA and DNA synthesis appeared to stop abruptly. In cells, the Clo DF13 factor continues to replicate during treatment with chloramphenicol long after chromosomal DNA synthesis ceases. When rifampin was included during chloramphenicol treatment of cells, synthesis of Clo DF13 plasmid DNA was blocked completely. Isolated, supercoiled Clo DF13 DNA, synthesized in cells or minicells in the presence of chloramphenicol, appeared to be sensitive to ribonuclease and alkali treatment. These treatments convert a relatively large portion of the covalently closed Clo DF13 DNA to the open circular form, whereas supercoiled Clo DF13 DNA, isolated from non-chloramphenicol-treated cells or minicells, is not significantly affected by these treatments. These results indicate that RNA synthesis and specifically Clo DF13 RNA synthesis are involved in Clo DF13 DNA replication and that the covalently closed Clo DF13 DNA, synthesized in the presence of chloramphenicol, contains one or more RNA sequences. De novo synthesis of chromosomal and Clo DF13-specific proteins is not required for the replication of the Clo DF13 factor. Supercoiled Clo DF13 DNA, isolated from a polA107 (Clo DF13) strain which lacks the 5' --> 3' exonucleolytic activity of DNA polymerase I, is insensitive to ribonuclease or alkali treatment, indicating that in this mutant the RNA sequences are still removed from the RNA-DNA hybrid.     

Thermal enhancement of the effectiveness of gamma radiation for induction of reproductive death in cultured mammalian cells.

The induction by gamma radiation of reproductive death in cultured cells derived from a rat ureter carcinoma (RUC-2) and from Chinese-hamster lung tissue (CH-V79) was shown to be enhanced by hyperthermic treatments at 41, 43 and 45 degrees C. The degree of enhancement was found to depend on the line of cells studied, the temperature employed and the level of damage considered. The influence of accumulating sublethal damage was decreased by hyperthermia, and the final slope of the radiation survival curve was increased. The degree of enhancement of lethal damage was found to depend on the time interval between the heat treatment and irradiation, especially at 41 degrees C.     

INK4a-deficient human diploid fibroblasts are resistant to RAS-induced senescence

The CDKN2A tumour suppressor locus encodes two distinct proteins, p16(INK4a) and p14(ARF), both of which have been implicated in replicative senescence, the state of permanent growth arrest provoked in somatic cells by aberrant proliferative signals or by cumulative population doublings in culture. Here we describe primary fibroblasts from a member of a melanoma-prone family who is homozygous for an intragenic deletion in CDKN2A. Analyses of the resultant gene products imply that the cells are p16(INK4a) deficient but express physiologically relevant levels of a frameshift protein that retains the known functions of p14(ARF). Although they have a finite lifespan, the cells are resistant to arrest by oncogenic RAS. Indeed, ectopic expression of RAS and telomerase (hTERT) results in outgrowth of anchorage-independent colonies that have essentially diploid karyotypes and functional p53. We find that in human fibroblasts, ARF is not induced demonstrably by RAS, pointing to significant differences between the proliferative barriers implemented by the CDKN2A locus in different cell types or species.     

Life-stage-specific differences in exploitation of food mixtures: diet mixing enhances copepod egg production but not juvenile development

Development, egg production and hatching success of the calanoidcopepods Temora longicornis and Pseudocalanus elongatus weremeasured in food mixtures to test their ability to obtain acomplete nutrition by combining different nutritionally poorfood species. In all the food mixtures used, the copepods failedto moult past the first copepodite stage, and the mortalitywas high. In sharp contrast, mixing two nutritionally poor foodspecies often resulted in egg production which was not significantlydifferent from nutritionally high quality food, although hatchingsuccess in many mixtures was low. Whereas egg production wassignificantly correlated with particulate organic nitrogen inthe diet, and independent of the highly unsaturated fatty acids(HUFAs), eicosapentaenoic acid (EPA) and docosahexaenoic acid(DHA), hatching increased with increasing DHA and EPA concentration.Growth and juvenile mortality were, however, independent ofeither nitrogen or HUFAs in the diet. Our results show thatadult copepods are effective in combining their nutrition fromseveral food sources, whereas juveniles are not. We suggestthat there are species- and life-stage-specific differencesin nutritional requirements and/or in the ability to digestand/or assimilate essential nutrients from food mixtures, whichmay significantly contribute to the success of copepod populationsin nature.     

Deriving Animal Behaviour from High-Frequency GPS: Tracking Cows in Open and Forested Habitat

The increasing spatiotemporal accuracy of Global Navigation Satellite Systems (GNSS) tracking systems opens the possibility to infer animal behaviour from tracking data. We studied the relationship between high-frequency GNSS data and behaviour, aimed at developing an easily interpretable classification method to infer behaviour from location data. Behavioural observations were carried out during tracking of cows (Bos Taurus) fitted with high-frequency GPS (Global Positioning System) receivers. Data were obtained in an open field and forested area, and movement metrics were calculated for 1 min, 12 s and 2 s intervals. We observed four behaviour types (Foraging, Lying, Standing and Walking). We subsequently used Classification and Regression Trees to classify the simultaneously obtained GPS data as these behaviour types, based on distances and turning angles between fixes. GPS data with a 1 min interval from the open field was classified correctly for more than 70% of the samples. Data from the 12 s and 2 s interval could not be classified successfully, emphasizing that the interval should be long enough for the behaviour to be defined by its characteristic movement metrics. Data obtained in the forested area were classified with a lower accuracy (57%) than the data from the open field, due to a larger positional error of GPS locations and differences in behavioural performance influenced by the habitat type. This demonstrates the importance of understanding the relationship between behaviour and movement metrics, derived from GNSS fixes at different frequencies and in different habitats, in order to successfully infer behaviour. When spatially accurate location data can be obtained, behaviour can be inferred from high-frequency GNSS fixes by calculating simple movement metrics and using easily interpretable decision trees. This allows for the combined study of animal behaviour and habitat use based on location data, and might make it possible to detect deviations in behaviour at the individual level.     

The Dutch N-cascade in the European perspective

The Netherlands is "well known" for its nitrogen problems; it has one of the highest reactive nitrogen (Nr) emission densities in the world. It is a small country at the delta of several large European rivers. Ever since the industrial revolution, there has been a growing excess of nutrients and related emissions into the atmosphere (ammonia, nitrogen oxides and nitrous oxide)and into groundwater and surface water (nitrate), leading to a large range of cascading environmental impacts. Vehicular traffic, sewage and animal husbandry are the main sources of oxidized and reduced forms of Nr. This paper provides an overview of the origin and fate of nitrogen in the Netherlands, the various reported impacts of nitrogen, the Dutch and European policies to reduce nitrogen emissions and related impacts. In addition, ways are presented to go forward to potentially solve the problems in a European perspective. Solutions include the improvement of nitrogen efficiencies in different systems, technological options and education.