A Protein Kinase-Dependent Block to Reinitiation of DNA Replication in G2 Phase in Mammalian Cells

Eukaryotic cells normally replicate their DNA only once between mitoses. Unlike G1 nuclei, intact G2 nuclei do not replicate during incubation inXenopusegg extract. However, artificial permeabilization of the nuclear membrane of G2 nuclei allows induction of new initiations byXenopusegg extract. This is consistent with the action of a replication licensing factor which is believed to enter the nucleus when the nuclear membrane breaks down at mitosis. Here, we show that G2 nuclei will initiate a new round of replication in the absence of nuclear membrane permeabilization, if they are preexposed to protein kinase inhibitorsin vivo.Competence to rereplicate is generated within 30 min of drug treatment, well before the scheduled onset of mitosis. This demonstrates that a protein kinase-dependent mechanism is continually active in G2 phase to actively prevent regeneration of replication capacity in mammalian cells. Kinase inhibition in G2 cells causes nuclear accumulation of replication protein A. Rereplication of kinase-inhibited G2 nuclei also depends on factors supplied byXenopusegg extract, which are distinct from those required for replication licensing.     

Genetic manipulation of 6-phosphofructokinase in potato tubers

The aim of this work was to discover whether genetic manipulation of 6-phosphofructokinase [EC 2.7.1.11; PFK(ATP)] influenced the rate of respiration of tuber tissue of Solanum tuberosum L. Transgenic plants were produced that contained the coding sequence of the Escherichia coli pfkA gene linked to a patatin promoter. Expression of this chimaeric gene in tubers resulted in a 14to 21-fold increase in the maximum catalytic activity of PFK(ATP) without affecting the activities of the other glycolytic enzymes. Tubers, and aged disks of tuber tissue, from transformed plants showed no more than a 30% fall in the content of hexose 6-monophosphates; the other intermediates of glycolysis increased threeto eightfold. Fructose-2,6-bisphosphate was barely detectable in aged disks of transformed tubers. The relative rates of ¹⁴CO₂ production from [1-¹⁴C]-and [6-¹⁴C]-glucose supplied to disks of transformed and control tubers were similar. Oxygen uptake and CO₂ production by aged disks of transformed tubers did not differ significantly from those from control tubers. The same was true of CO₂ production, in air, and in nitrogen, for tuber tissue. It is concluded that PFK(ATP) does not dominate the control of respiration in potato tubers.Abbreviations Fru2,6bisP fructose-2,6-bisphosphate - FW freshweight - GUS -glucuronidase - PFK(ATP) 6-phosphofructokinase - PFK(PPi) pyrophosphate: fructose-6-phosphate 1-phosphotransferase     

Separation of v-Src-induced mitogenesis and morphological transformation by inhibition of AP-1.

v-Src activity results in both morphological transformation and reentry of quiescent chick embryo fibroblasts (CEF) into cell cycle. We have previously used temperature-sensitive v-Src mutants to show that enhanced activity of cellular AP-1 in the first few hours after activation of v-Src invariably precedes the biological consequences. Here we have investigated whether the early activation of AP-1 is essential for any or all of the v-Src responses by using a mutant c-Fos that comprises the leucine zipper and a disrupted basic region. Expression of the c-Fos mutant partially reduced cellular AP-1 activity in exponentially growing cells. However, in CEF that had been made quiescent by serum deprivation, v-Src-induced stimulation of AP-1 DNA binding activity was substantially reduced. In addition, quiescent CEF stably transfected with this mutant show an impaired mitogenic response to v-Src, indicating that the AP-1 stimulation is a necessary prerequisite for cell-cycle reentry. The ability of v-Src to morphologically transform quiescent CEF was not impaired by the inhibition of AP-1 stimulation, indicating that the mitogenic and morphological consequences of v-Src have distinguishable biochemical mediators. Focal adhesion kinase, a recently identified determinant of cell morphology, undergoes a gel mobility shift, characteristic of its hyperphosphorylated state, in response to v-Src activation in cells expressing the inhibitory AP-1 protein. This provides further evidence that the pathways that regulate morphological transformation are independent of AP-1.     

Cell volume measurement as an estimation of mammalian cell biomass

Measurements of volume distributions and dry weight are made on hybridoma cells in culture. The volume of viable hybridoma cells is significantly larger than that of nonviable cells. During exponential growth, the volume of the viable hybridoma cells is found to be significantly larger than that during other stages of batch culture. Proportionality is found between the volume of the cells and their dry weight, indicating that the volume data can be used in conjunction with cell concentration data as a practical technique for indirect measurement of the biomass concentration present in a culture. Comparison of dry weight concentrations in continuous culture to predictions from the volume data shows very good agreement.     

Exploring movement patterns and changing distributions of baleen whales in the western North Atlantic using a decade of passive acoustic data

Six baleen whale species are found in the temperate western North Atlantic Ocean, with limited information existing on the distribution and movement patterns for most. There is mounting evidence of distributional shifts in many species, including marine mammals, likely because of climate‐driven changes in ocean temperature and circulation. Previous acoustic studies examined the occurrence of minke (Balaenoptera acutorostrata) and North Atlantic right whales (NARW; Eubalaena glacialis). This study assesses the acoustic presence of humpback (Megaptera novaeangliae), sei (B. borealis), fin (B. physalus), and blue whales (B. musculus) over a decade, based on daily detections of their vocalizations. Data collected from 2004 to 2014 on 281 bottom‐mounted recorders, totaling 35,033 days, were processed using automated detection software and screened for each species' presence. A published study on NARW acoustics revealed significant changes in occurrence patterns between the periods of 2004–2010 and 2011–2014; therefore, these same time periods were examined here. All four species were present from the Southeast United States to Greenland; humpback whales were also present in the Caribbean. All species occurred throughout all regions in the winter, suggesting that baleen whales are widely distributed during these months. Each of the species showed significant changes in acoustic occurrence after 2010. Similar to NARWs, sei whales had higher acoustic occurrence in mid‐Atlantic regions after 2010. Fin, blue, and sei whales were more frequently detected in the northern latitudes of the study area after 2010. Despite this general northward shift, all four species were detected less on the Scotian Shelf area after 2010, matching documented shifts in prey availability in this region. A decade of acoustic observations have shown important distributional changes over the range of baleen whales, mirroring known climatic shifts and identifying new habitats that will require further protection from anthropogenic threats like fixed fishing gear, shipping, and noise pollution.