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     

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.     

Akt-Induced Phosphorylation of N-CoR at Serine 1450 Contributes to Its Misfolded Conformational Dependent Loss (MCDL) in Acute Myeloid Leukemia of the M5 Subtype

The nuclear receptor co-repressor (N-CoR) is a key component of the generic co-repressor complex that plays an important role in the control of cellular growth and differentiation. As shown by us recently, the growth suppressive function of N-CoR largely relies on its capacity to repress Flt3, a key regulator of cellular gorwth during normal and malignant hematopoesis. We further demonstrated how de-repression of Flt3 due to the misfolded conformation dependent loss (MCDL) of N-CoR contributed to malignant growth in acute myeloid leukemia (AML). However, the molecular mechanism underlying the MCDL of N-CoR and its implication in AML pathogenesis is not fully understood. Here, we report that Akt-induced phosphorylation of N-CoR at the consensus Akt motif is crucial for its misfolding and subsequent loss in AML (AML-M5). N-CoR displayed significantly higher level of serine specific phosphorylation in almost all AML-M5 derived cells and was subjected to processing by AML-M5 specific aberrant protease activity. To identify the kinase linked to N-CoR phosphorylation, a library of activated kinases was screened with the extracts of AML cells; leading to the identification of Akt as the putative kinase linked to N-CoR phosphorylation. Consistent with this finding, a constitutively active Akt consistently phosphorylated N-CoR leading to its misfolding; while the therapeutic and genetic ablation of Akt largely abrogated the MCDL of N-CoR in AML-M5 cells. Site directed mutagenic analysis of N-CoR identified serine 1450 as the crucial residue whose phosphorylation by Akt was essential for the misfolding and loss of N-CoR protein. Moreover, Akt-induced phosphorylation of N-CoR contributed to the de-repression of Flt3, suggesting a cross talk between Akt signaling and N-CoR misfolding pathway in the pathogenesis of AML-M5. The N-CoR misfolding pathway could be the common downstream thread of pleiotropic Akt signaling activated by various oncogenic insults in some subtypes of leukemia and solid tumors.     

Plasmodium falciparum Erythrocytic Stage Parasites Require the Putative Autophagy Protein PfAtg7 for Normal Growth

Analysis of the Plasmodium falciparum genome reveals a limited number of putative autophagy genes, specifically the four genes involved in ATG8 lipidation, an essential step in formation of autophagosomes. In yeast, Atg8 lipidation requires the E1-type ligase Atg7, an E2-type ligase Atg3, and a cysteine protease Atg4. These four putative P. falciparum ATG (PfATG) genes are transcribed during the parasite’s erythrocytic stages. PfAtg7 has relatively low identity and similarity to yeast Atg7 (14.7% and 32.2%, respectively), due primarily to long insertions typical of P. falciparum. Excluding the insertions the identity and similarity are higher (38.0% and 70.8%, respectively). This and the fact that key residues are conserved, including the catalytic cysteine and ATP binding domain, we hypothesize that PfAtg7 is the activating enzyme of PfAtg8. To assess the role of PfAtg7 we have generated two transgenic parasite lines. In one, the PfATG7 locus was modified to introduce a C-terminal hemagglutinin tag. Western blotting reveals two distinct protein species, one migrating near the predicted 150 kDa and one at approximately 65 kDa. The second transgenic line introduces an inducible degradation domain into the PfATG7 locus, allowing us to rapidly attenuate PfAtg7 protein levels. Corresponding species are also observed in this parasite line at approximately 200 kDa and 100 kDa. Upon PfATG7 attenuation parasites exhibit a slow growth phenotype indicating the essentiality of this putative enzyme for normal growth.     

Model vs. playback experiments: The impact of sensory mode on predator-specific escape responses in saki monkeys

Although experimentally simulating predator presence helps improve sample sizes in studies of free-ranging animals, few studies have examined whether auditory playbacks and visual models produce similar results. Additionally, it is unclear if anti-predator strategies are specific to predator hunting styles in understudied Neotropical pitheciid primates, limiting what we can generalize about this phenomenon across this taxonomic order. We conducted predator simulation experiments to assess whether wild Rylands' bald-faced saki monkeys (Pithecia rylandsi) recognize predators based solely on acoustic cues, exhibit predator-specific responses to different predator types, and vary responses to presentations in different sensory modes. In our playback experiments, sakis had weak responses to non-predator control vocalizations compared to jaguar growls and harpy eagle shrieks. In most predator playbacks, subjects' first glance corresponded to the direction from which simulated predators would typically attack (above vs. below). However, although sakis exhibited appropriate movement responses to harpy playbacks (i.e., descending canopy), they exhibited no clear movement patterns when presented with jaguar playbacks. In contrast, jaguar model experiments consistently elicited fast approaches, mobbing-style responses, and long alarm calling bouts. Thus, if we had relied on playbacks alone, we might have concluded that sakis have only generalized responses to terrestrial ambush predators. In fact, in all variables measured (e.g., latency, number of calls, and response duration), models of both predator species elicited stronger reactions than playbacks. Results indicate that bald-faced sakis can identify predators based solely on vocalizations, but do not exhibit predator-specific escape responses to terrestrial predators based on acoustic cues alone. The differential response to playbacks and models calls into question the reliability of using acoustic-only stimuli to assess the specificity of anti-predator behavior to predator hunting styles in some primate species.