Activation of PKR causes amyloid ß-peptide accumulation via de-repression of BACE1 expression

BACE1 is a key enzyme involved in the production of amyloid ß-peptide (Aß) in Alzheimer''s disease (AD) brains. Normally, its expression is constitutively inhibited due to the presence of the 5′untranslated region (5′UTR) in the BACE1 promoter. BACE1 expression is activated by phosphorylation of the eukaryotic initiation factor (eIF)2-alpha, which reverses the inhibitory effect exerted by BACE1 5′UTR. There are four kinases associated with different types of stress that could phosphorylate eIF2-alpha. Here we focus on the double-stranded (ds) RNA-activated protein kinase (PKR). PKR is activated during viral infection, including that of herpes simplex virus type 1 (HSV1), a virus suggested to be implicated in the development of AD, acting when present in brains of carriers of the type 4 allele of the apolipoprotein E gene. HSV1 is a dsDNA virus but it has genes on both strands of the genome, and from these genes complementary RNA molecules are transcribed. These could activate BACE1 expression by the PKR pathway. Here we demonstrate in HSV1-infected neuroblastoma cells, and in peripheral nervous tissue from HSV1-infected mice, that HSV1 activates PKR. Cloning BACE1 5′UTR upstream of a luciferase (luc) gene confirmed its inhibitory effect, which can be prevented by salubrinal, an inhibitor of the eIF2-alpha phosphatase PP1c. Treatment with the dsRNA analog poly (I∶C) mimicked the stimulatory effect exerted by salubrinal over BACE1 translation in the 5′UTR-luc construct and increased Aß production in HEK-APPsw cells. Summarizing, our data suggest that PKR activated in brain by HSV1 could play an important role in the development of AD.     

Biogeochemistry of Mariana Islands coastal sediments: terrestrial influence on /gd13, Ash,CaCO3, Al,Fe, Si and P

Stable C isotope ratios (¹³C-PDB), percentages of organic matter, and HCl insoluble ash and soluble carbonates, extractable Fe, Al, Si and P were used to determine the distribution and accumulation of terrestrial material in reef-flat moats and lagoons of two high islands (Guam and Saipan) in the western tropical Pacific. Carbonate sediments of a reef-flat moat infiltrated by seepage of aquifer waters (but without surface runoff) were depleted in both P (by 38%) and ¹³C (by 41%) and enriched in Si (by 100%) relative to offshore lagoon sediments. Iron and ash accumulated in depositional regimes regardless of the occurrence of runoff but was depleted from coarse-grained carbonates in turbulent regimes. Aluminum (>ca. 10 to 20 mol g^-1), Fe (>ca. 1 to 3 mol g^-1) and ash (>0.5%) indicated terrigenous influence which was corroborated by depletions in both ¹³C and P. Low-salinity geochemical segregation, natural biochemical accumulation, as well as long-shore currents and eddies help sequester these materials nearshore.     

Tallgrass prairie restoration: implications of increased atmospheric nitrogen deposition when site preparation minimizes adventive grasses

Site preparation designed to exhaust the soil seedbank of adventive species can improve the success of tallgrass prairie restoration. Despite these efforts, increased rates of atmospheric nitrogen (N) deposition over the next century could potentially promote the growth of nitrophilic, adventive species in tallgrass restoration projects. We used a field experiment to examine how N addition affected species composition and plant productivity over the first 3 years of a tallgrass prairie restoration that was preceded by the planting of glyphosate‐resistant crops and multiple applications of glyphosate to exhaust the pre‐existing seedbank. We predicted that N addition would increase the percent cover of adventive plant species not included in the original seeding. Contrary to our prediction, only the cover of native species increased with N addition; native non‐leguminous forbs increased substantially, with Conyza canadensis (a weedy native species not part of the restoration seed mix) exploiting the combination of high N and bare ground in the first year, and non‐leguminous forbs (in particular Monarda fistulosa) and native C₃ grasses, all of which were seeded, increasing with N addition by the third year. Native legumes was the only functional group that exhibited lower cover in N addition plots than in control plots. There was no significant response by native C₄ grasses to N addition, and adventive grasses remained mostly absent from the plots. Overall, our results suggest that site pre‐treatment with herbicide may continue to be effective in minimizing adventive grasses in restored tallgrass prairie, despite future increases in atmospheric N deposition.     

N-glycans on Nipah virus fusion protein protect against neutralization but reduce membrane fusion and viral entry

Nipah virus (NiV) is a deadly emerging paramyxovirus. The NiV attachment (NiV-G) and fusion (NiV-F) envelope glycoproteins mediate both syncytium formation and viral entry. Specific N-glycans on paramyxovirus fusion proteins are generally required for proper conformational integrity and biological function. However, removal of individual N-glycans on NiV-F had little negative effect on processing or fusogenicity and has even resulted in slightly increased fusogenicity. Here, we report that in both syncytium formation and viral entry assays, removal of multiple N-glycans on NiV-F resulted in marked increases in fusogenicity (>5-fold) but also resulted in increased sensitivity to neutralization by NiV-F-specific antisera. The mechanism underlying the hyperfusogenicity of these NiV-F N-glycan mutants is likely due to more-robust six-helix bundle formation, as these mutants showed increased fusion kinetics and were more resistant to neutralization by a fusion-inhibitory reagent based on the C-terminal heptad repeat region of NiV-F. Finally, we demonstrate that the fusogenicities of the NiV-F N-glycan mutants were inversely correlated with the relative avidities of NiV-F's interactions with NiV-G, providing support for the attachment protein "displacement" model of paramyxovirus fusion. Our results indicate that N-glycans on NiV-F protect NiV from antibody neutralization, suggest that this "shielding" role comes together with limiting cell-cell fusion and viral entry efficiencies, and point to the mechanisms underlying the hyperfusogenicity of these N-glycan mutants. These features underscore the varied roles that N-glycans on NiV-F play in the pathobiology of NiV entry but also shed light on the general mechanisms of paramyxovirus fusion with host cells.     

Expression of the p12 subunit of human DNA polymerase δ (Pol δ), CDK inhibitor p21WAF1, Cdt1, cyclin A,PCNA and Ki-67 in relation to DNA replication in individual cells

We recently reported that the p12 subunit of human DNA polymerase δ (Pol δ4) is degraded by CRL4^Cdt2 which regulates the licensing factor Cdt1 and p21^WAF1 during the G₁ to S transition. Presently, we performed multiparameter laser scanning cytometric analyses of changes in levels of p12, Cdt1 and p21^WAF1, detected immunocytochemically in individual cells, vis-à-vis the initiation and completion of DNA replication. The latter was assessed by pulse-labeling A549 cells with the DNA precursor ethynyl-2′-deoxyribose (EdU). The loss of p12 preceded the initiation of DNA replication and essentially all cells incorporating EdU were p12 negative. Completion of DNA replication and transition to G₂ phase coincided with the re-appearance and rapid rise of p12 levels. Similar to p12 a decline of p21^WAF1 and Cdt1 was seen at the end of G₁ phase and all DNA replicating cells were p21^WAF1 and Cdt1 negative. The loss of p21^WAF1 preceded that of Cdt1 and p12 and the disappearance of the latter coincided with the onset of DNA replication. Loss of p12 leads to conversion of Pol δ4 to its trimeric form, Pol δ3, so that the results provide strong support to the notion that Pol δ3 is engaged in DNA replication during unperturbed progression through the S phase of cell cycle. Also assessed was a correlation between EdU incorporation, likely reflecting the rate of DNA replication in individual cells, and the level of expression of positive biomarkers of replication cyclin A, PCNA and Ki-67 in these cells. Of interest was the observation of stronger correlation between EdU incorporation and expression of PCNA (r = 0.73) than expression of cyclin A (r = 0.47) or Ki-67 (r = 0.47).     

Development of pheromone-based mating disruption for control of the eye-spotted bud moth, Spilonota ocellana

The potential for pheromone-based mating disruption of eye-spotted bud moth (ESBM), Spilonota ocellana (Denis & Schiffermüller) (Lepidoptera: Tortricidae), in apple orchards in the Okanagan Valley of British Columbia was examined in small-plot trials. In a preliminary experiment, treatment of the orchard atmosphere with a 99:1 blend of Z8-tetradecenyl acetate (Z8-14:OAc) and Z8-tetradecenyl alcohol (Z8-14:OH) completely inhibited captures of male ESBM in pheromone-baited traps, but treatment with Z8-14:OAc alone did not. Therefore, all subsequent trials used the two-component blend as a disruptant. Mean catches of male ESBM in traps baited with 0.1, 1, 10, or 20 mg of 99:1 Z8-14:OAc and Z8-14:OH were significantly reduced (81–97%) in pheromone-treated plots relative to similar traps placed in control plots. In both control and treated plots, there was a significant positive relationship between trap bait dose and trap catch. In pheromone-treated plots, this suggests that high doses of trap baits over-ride the camouflage effect of disruption or overcome the effect of sensory adaptation and habituation. The number of virgin-female baited traps capturing at least one male ESBM was reduced by 96%, and mating of virgin females on mating tables was reduced by 95% in plots treated with the two-component pheromone. The total amount of Z8-14:OAc released from pheromone disruption dispensers during the latter field trials was estimated to average 6.4 g ha^-1 over 11 days or 26.4 mg ha^-1 h^-1. The low levels of Z8-14:OH released from disruption dispensers could not be estimated by GC analysis. Dispensers loaded with a 99:1 blend of Z8-14:OAc and Z8-14:OH were highly attractive to males in baited traps, indicating that they have the potential to induce false trail following.     

Partitioning seasonal time: interactions among size, foraging activity and diet in leaf-litter frogs

This study investigates hypotheses about partitioning of food resources among all species and several size classes in an assemblage of diurnal leaf-litter frogs in central Amazonia. All species in this assemblage change the type and size of prey as they grow. An ordination of diet composition was significantly associated with frog size and species-specific behaviour. However, a partial Mantel analysis indicated that species explained about 1.5 times more of the variation in diet overlap between individuals than frog size. Diet and foraging activity are correlated in juveniles, but not in adults, and this result holds whether species are considered as statistically independent observations or whether relationships are analysed using phylogenetically independent contrasts. This study showed that the partitioning of food resources between species changes with the population size structures. Thus, intraspecific and interspecific changes in diet, coupled with different patterns of juvenile recruitment, cause diet segregation among species due to temporal segregation of equivalent size classes. Received: 25 August 1997 / Accepted: 1 April 1998     

Cardioprotection involves activation of NF-kappa B via PKC-dependent tyrosine and serine phosphorylation of I kappa B-alpha

Previous studies indicated that activation of PKC and Src tyrosine kinases by ischemic preconditioning (PC) may participate in the activation of NF-kappa B. However, the molecular mechanisms underlying activation of NF-kappa B during ischemic PC remain unknown. In the hearts of conscious rabbits, it was found that ischemic PC (6 cycles of 4-min coronary occlusion and 4-min reperfusion) significantly induced both tyrosine (+226.9 +/- 42%) and serine (+137.0 +/- 36%) phosphorylation of the NF-kappa B inhibitory protein I kappa B-alpha, concomitant with increased activation of the I kappa B-alpha kinases IKK alpha (+255.0 +/- 46%) and IKK beta (+173.1 +/- 35%). Furthermore, both tyrosine and serine phosphorylation of I kappa B-alpha were blocked by pretreatment with either the nonreceptor tyrosine kinase inhibitor lavendustin-A (LD-A) or the PKC inhibitor chelerythrine (Che) (both given at doses previously shown to block ischemic PC). Interestingly, Che completely abolished PC-induced activation of IKK alpha/beta, whereas LD-A had no effect. In addition, I kappa B-alpha protein level did not change during ischemic PC. Together, these data indicate that ischemic PC-induced activation of NF-kappa B occurs through both tyrosine and serine phosphorylation of I kappa B-alpha and is regulated by nonreceptor tyrosine kinases and PKC.     

A potent mechanism-inspired O-GlcNAcase inhibitor that blocks phosphorylation of tau in vivo

Pathological hyperphosphorylation of the microtubule-associated protein tau is characteristic of Alzheimer's disease (AD) and the associated tauopathies. The reciprocal relationship between phosphorylation and O-GlcNAc modification of tau and reductions in O-GlcNAc levels on tau in AD brain offers motivation for the generation of potent and selective inhibitors that can effectively enhance O-GlcNAc in vertebrate brain. We describe the rational design and synthesis of such an inhibitor (thiamet-G, K(i) = 21 nM; 1) of human O-GlcNAcase. Thiamet-G decreased phosphorylation of tau in PC-12 cells at pathologically relevant sites including Thr231 and Ser396. Thiamet-G also efficiently reduced phosphorylation of tau at Thr231, Ser396 and Ser422 in both rat cortex and hippocampus, which reveals the rapid and dynamic relationship between O-GlcNAc and phosphorylation of tau in vivo. We anticipate that thiamet-G will find wide use in probing the functional role of O-GlcNAc in vertebrate brain, and it may also offer a route to blocking pathological hyperphosphorylation of tau in AD.