Regulation of ploidy and senescence by the AMPK‐related kinase NUAK1

Senescence is an irreversible cell‐cycle arrest that is elicited by a wide range of factors, including replicative exhaustion. Emerging evidences suggest that cellular senescence contributes to ageing and acts as a tumour suppressor mechanism. To identify novel genes regulating senescence, we performed a loss‐of‐function screen on normal human diploid fibroblasts. We show that downregulation of the AMPK‐related protein kinase 5 (ARK5 or NUAK1) results in extension of the cellular replicative lifespan. Interestingly, the levels of NUAK1 are upregulated during senescence whereas its ectopic expression triggers a premature senescence. Cells that constitutively express NUAK1 suffer gross aneuploidies and show diminished expression of the genomic stability regulator LATS1, whereas depletion of NUAK1 with shRNA exerts opposite effects. Interestingly, a dominant‐negative form of LATS1 phenocopies NUAK1 effects. Moreover, we show that NUAK1 phosphorylates LATS1 at S464 and this has a role in controlling its stability. In summary, our work highlights a novel role for NUAK1 in the control of cellular senescence and cellular ploidy.     

Artificially increased ascorbate content affects zeaxanthin formation but not thermal energy dissipation or degradation of antioxidants during cold-induced photooxidative stress in maize leaves

Infiltrating detached maize (Zeamays L.) leaves with L-galactono-1,4-lactone (L-GAL) resulted in a 4-fold increase in the content of leaf ascorbate. Upon exposure to high irradiance (1000 μmol photons m⁻² s⁻¹) at 5 °C, L-GAL leaves de-epoxidized the xanthophyll-cycle pigments faster than the control leaves; the maximal ratio of de-epoxidized xanthophyll-cycle pigments to the whole xanthophyll-cycle pool was the same in both leaf types. The elevated ascorbate content, together with the faster violaxanthin de-epoxidation, did not affect the degree of photoinhibition and the kinetics of the recovery from photoinhibition, assayed by monitoring the maximum quantum efficiency of photosystem II primary photochemistry (F_v/F_m). Under the experimental conditions, the thermal energy dissipation seems to be zeaxanthin-independent since, in contrast to the de-epoxidation, the decrease in the efficiency of excitation-energy capture by open photosystem II reaction centers (F_v′/F_m′) during the high-irradiance treatment at low temperature showed the same kinetic in both leaf types. This was also observed for the recovery of the maximal fluorescence after stress. Furthermore, the elevated ascorbate content did not diminish the degradation of pigments or α-tocopherol when leaves were exposed for up to 24 h to high irradiance at low temperature. Moreover, a higher content of ascorbate appeared to increase the requirement for reduced glutathione. Received: 20 May 1999 / Accepted: 29 October 1999     

The Equivocal Relationship Between Territoriality and Scent Marking in Wild Saddleback Tamarins (<Emphasis Type="Italic">Saguinus fuscicollis</Emphasis>)

Researchers have often assumed that scent marking serves a territorial function in callitrichines, although some controversy exists. To fulfill such a function, scent marks should 1) prevent intrusions, 2) ensure access to feeding resources, 3) enable avoidance of intergroup encounters, or 4) play an important role in the aggressive encounters between groups. We studied 13 saddleback tamarins (Saguinus fuscicollis) belonging to 3 free-ranging groups, which formed mixed-species troops with moustached tamarins (S. mystax) in the Amazonian rain forest of Peru. None of the predictions were confirmed. The tamarins used a border-marking strategy, marking more on the periphery of their territory. However, feeding trees in overlap and encounter areas received more scent marking but were still visited by neighboring groups. Intergroup encounters occurred more often than expected, and scent-marking frequency was not higher during them than when no other group was present. It appears that instead of defending a territory in the classic sense, the tamarins are optimizing signal transmission by depositing their scents where the probability of detection by neighbors is higher. Saddleback tamarins may use shared areas of their home ranges to exchange information with neighboring groups, perhaps regarding reproductive opportunities.     

Restoration of human dystrophin following transplantation of exon-skipping-engineered DMD patient stem cells into dystrophic mice

Duchenne muscular dystrophy (DMD) is a hereditary disease caused by mutations that disrupt the dystrophin mRNA reading frame. In some cases, forced exclusion (skipping) of a single exon can restore the reading frame, giving rise to a shorter, but still functional, protein. In this study, we constructed lentiviral vectors expressing antisense oligonucleotides in order to induce an efficient exon skipping and to correct the initial frameshift caused by the DMD deletion of CD133+ stem cells. The intramuscular and intra-arterial delivery of genetically corrected CD133 expressing myogenic progenitors isolated from the blood and muscle of DMD patients results in a significant recovery of muscle morphology, function, and dystrophin expression in scid/mdx mice. These data demonstrate that autologous engrafting of blood or muscle-derived CD133+ cells, previously genetically modified to reexpress a functional dystrophin, represents a promising approach for DMD.     

The M‐type receptor PLA2R regulates senescence through the p53 pathway

Senescence is a stable proliferative arrest induced by various stresses such as telomere erosion, oncogenic or oxidative stress. Compelling evidence suggests that it acts as a barrier against tumour development. Describing new mechanisms that favour an escape from senescence can thus reveal new insights into tumorigenesis. To identify new genes controlling the senescence programme, we performed a loss‐of‐function genetic screen in primary human fibroblasts. We report that knockdown of the M‐type receptor PLA2R (phospholipase A2 receptor) prevents the onset of replicative senescence and diminishes stress‐induced senescence. Interestingly, expression of PLA2R increases during replicative senescence, and its ectopic expression results in premature senescence. We show that PLA2R regulates senescence in a reactive oxygen species–DNA damage–p53‐dependent manner. Taken together, our study identifies PLA2R as a potential new tumour suppressor gene crucial in the induction of cellular senescence through the activation of the p53 pathway.     

Discovery and characterization of novel indole and 7-azaindole derivatives as inhibitors of β-amyloid-42 aggregation for the treatment of Alzheimer’s disease

The aggregation of amyloid-β peptides into cytotoxic oligomeric and fibrillary aggregates is believed to be one of the major pathological events in Alzheimer disease. Here we report the design and synthesis of a novel series of indole and 7-azaindole derivatives containing, nitrile, piperidine and N-methyl-piperidine substituents at the 3-position to prevent the pathological self-assembly of amyloid-β. We have further demonstrated that substitution of the azaindole and indole derivatives at the 3 positions is required to obtain compounds with improved physicochemical properties to allow brain penetration.     

Acute exposure to 50 Hz magnetic field does not affect hematologic or immunologic functions in healthy young men: A circadian study

Some epidemiological studies report a relationship between magnetic field exposure and such human diseases as leukemia and immune system disturbances. The few published studies on animals do not demonstrate field exposure-related alterations in hematologic and immune systems. The data presented here are part of a broader study designed to investigate the possible effects of acute exposure to a 50 Hz linearly polarized magnetic field (10 μT) on hematologic and immunologic functions. Thirty-two young men (20–30 years old) were divided into two groups (control group, i.e., sham-exposed, 16 subjects; exposed group, 16 subjects). All subjects participated in two 24 h experiments to evaluate the effects of both continuous and intermittent (1 h “off” and 1 h with the field switched “on” and “off” every 15 s) exposure to linearly polarized magnetic fields. The subjects were exposed to the magnetic field (generated by three Helmholtz coils per bed) from 23:00 to 08:00 while lying down. Blood samples were collected during each session at 3 h intervals from 11:00 to 20:00 and hourly from 22:00 to 08:00. No significant differences were observed between sham-exposed (control) and exposed men for hemoglobin concentration, hematocrit, red blood cells, platelets, total leukocytes, monocytes, lymphocytes, eosinophils, or neutrophils. Immunologic variables [CD3, CD4, CD8, natural killer (NK) cells and B cells] were unaltered. To our knowledge, this study is the first to document the effects of a 50 Hz magnetic field on the circadian rhythm of human hematologic and immune functions, and it suggests that acute exposure to either a continuous or an intermittent 50 Hz linearly polarized magnetic field of 10 μT, at least under the conditions of our experiment, does not affect either these functions or their circadian rhythms in healthy young men. © 1996 Wiley-Liss, Inc.     

Influence of electromagnetic fields emitted by GSM-900 cellular telephones on the circadian patterns of gonadal, adrenal and pituitary hormones in men

The potential health risks of radiofrequency electromagnetic fields (RF EMFs) emitted by mobile phones are currently of considerable public interest. The present study investigated the effect of exposure to 900 MHz GSM radiofrequency radiation on steroid (cortisol and testosterone) and pituitary (thyroid-stimulating hormone, growth hormone, prolactin and adrenocorticotropin) hormone levels in 20 healthy male volunteers. Each subject was exposed to RF EMFs through the use of a cellular phone for 2 h/day, 5 days/ week, for 4 weeks. Blood samples were collected hourly during the night and every 3 h during the day. Four sampling sessions were performed at 15-day intervals: before the beginning of the exposure period, at the middle and the end of the exposure period, and 15 days later. Parameters evaluated included the maximum serum concentration, the time of this maximum, and the area under the curve for hormone circadian patterns. Each individual's pre-exposure hormone concentration was used as his control. All hormone concentrations remained within normal physiological ranges. The circadian profiles of prolactin, thyroid-stimulating hormone, adrenocorticotropin and testosterone were not disrupted by RF EMFs emitted by mobile phones. For growth hormone and cortisol, there were significant decreases of about 28% and 12%, respectively, in the maximum levels when comparing the 2-week (for growth hormone and cortisol) and 4-week (for growth hormone) exposure periods to the pre-exposure period, but no difference persisted in the postexposure period. Our data show that the 900 MHz EMF exposure, at least under our experimental conditions, does not appear to affect endocrine functions in men.     

Effects of Bacillus thuringiensis δ-Endotoxins on the Pea Aphid (Acyrthosiphon pisum)

Four Bacillus thuringiensis δ-endotoxins, Cry3A, Cry4Aa, Cry11Aa, and Cyt1Aa, were found to exhibit low to moderate toxicity on the pea aphid, Acyrthosiphon pisum, in terms both of mortality and growth rate. Cry1Ab was essentially nontoxic except at high rates. To demonstrate these effects, we had to use exhaustive buffer-based controls.Many species of aphids are important sucking-insect pests that feed on plant vascular fluids. Their feeding mechanism makes these insects excellent vectors for many plant pathogens, especially viruses, yet less amenable to standard, nonsystemic chemical control by insecticides. Minor effects on the survival and fecundity of aphids reared on Bacillus thuringiensis (Bt) crops have been noted in some studies but not in others (1, 3, 6). However, the sensitivity of aphids to Bt toxins, or the lack thereof, has not been previously tested through artificial-diet bioassays with exhaustive buffer-based controls.Bt δ-endotoxins Cyt1A, Cry4A/Cry4B, and Cry11, obtained from three recombinant strains of B. thuringiensis subsp. israelensis, as well as Cry1Ab and Cry3A, obtained from recombinant Escherichia coli, were purified by ultracentrifugation in a discontinuous sucrose gradient as described previously (9). Cry proteins were solubilized in solubilization buffer (50 mM Na₂CO₃, 100 mM NaCl, pH 10) with dithiothreitol (10 mM) added before use. Cyt1A was first solubilized on 10 mM Na₂CO₃ (pH 11) buffer and then neutralized at pH 7.5 to 8 with 10 μl HCl (1 N). Both solubilized and trypsin-digested samples (1:30 over toxin weight) were used at different concentrations (32, 125, and 500 μg/ml; trypsin-activated toxin concentrations were calculated on the basis of the preactivation concentrations of the protoxins) to supplement the AP3 aphid synthetic diet (7) used to feed Acyrthosiphon pisum (LL01 green clone). Ampicillin (100 μg/ml), an ineffective antibiotic for A. pisum or its obligate symbiont Buchnera, was added to the medium to avoid bacterial growth. For each concentration, 30 nymphs (10 nymphs/box and three repetitions) were bioassayed at 20°C and under a 16:8 (light-dark) photoperiod. Survival time was calculated from aphid deposition on the test diet (day 0). Mortality was surveyed daily, and body weights of survivors were noted at day 7. ST₅₀ (median survival time after challenge) was calculated by using an actuarial survival analysis (Statview) with censoring values of survivors at the end of the experiments. The approximate concentrations resulting in a 50% decrease in mean body weight (IC₅₀) and killing of 50% of the insects tested (LC₅₀) were calculated at the end of the experiments from the growth reduction and mortality data, respectively, derived with the three doses by using Statview and the censoring values of survivors.All of the Cry δ-endotoxins tested were lethal to A. pisum and retarded the growth of survivors (Fig. ​(Fig.11 and ​and2).2). Mortalities ranged from only 25% (Cry1Ab) to 100% (Cry4 and Cry11) after 3 to 6 days of exposure to 500 μg/ml of solubilized protein (Fig. ​(Fig.1).1). When significant mortalities were achieved (Cry3A, Cry4, and Cry11), trypsin activation enhanced toxicity. Activation of Cry4 at the intermediate concentration tested (125 μg/ml) resulted in a twofold increase in mortality (Fig. ​(Fig.1D).1D). ST₅₀s were calculated for both solubilized protoxins and activated Cry3A, Cry4, and Cry11. The ST₅₀s (at 500 μg/ml) ranged from 1.8 ± 0.14 days for solubilized Cry4 and Cry11 to 3.7 ± 1.2 days for trypsin-activated Cry3A (Table ​(Table1).1). Control aphids fed buffer all survived for >8 days. The LC₅₀ of Cry1Ab was not calculated, since mortality associated with Cry1Ab reached a plateau at 500 μg/ml. The LC₅₀ of Cry4 was estimated to be 70 to 100 μg/ml (data not shown).Open in a separate windowFIG. 1.Mortality assays over the nymphal life stage of the pea aphid, A. pisum, upon ingestion of artificial diets containing purified Bt toxins after either solubilization (open symbols) or solubilization and trypsin activation (solid symbols). The toxins used were Cry1Ab (circles), Cry3A (squares), a mixture of Cry4A and Cry4B (diamonds), and Cry11A (triangles). The soluble-toxin doses used were low at 32 μg/ml (blue), intermediate at 125 μg/ml (violet), and high at 500 μg/ml (red). Assays were carried out with 30 initial neonate insects in three batches of 10 individuals.Open in a separate windowFIG. 2.Growth inhibition assays with purified Bt toxins Cry3A, Cry4, and Cry 11 (A) and Cry1Ab and Cyt1A (B) on the pea aphid, A. pisum. Toxins were added to the diet either after solubilization (open symbols) or after solubilization and trypsin activation (solid symbols). Error bars show the standard errors (SE) of individual weights at day 7 of experiments, standardized by the control group mean weight (toxin dose, 0; initial number, 30). Color coding of toxins: Cry3A, red squares; Cry4A and Cry4B mixture, violet diamonds; Cry11A, blue triangles; Cry1Ab, green circles; Cyt1A, yellow squares. In the experiment with Cry1Ab (B), the toxin was purified by high-performance liquid chromatography and activated toxin was provided as a salt-free lyophilisate by W. Moar (Auburn University, Auburn, AL).

TABLE 1.

ST₅₀s of pea aphids feeding on solubilized Cry toxins and solubilized Cry toxins activated with trypsin