Self-Assembly of a U(VI)-Containing Polytungstate Tetramer with Lewis Acid-Base Catalytic Activity for a Dehydration Condensation Reaction.

A novel U(VI)-containing polytungstate (U-POW) tetramer, {K1.37Na26.63[K2(UO2)4Cl0.5(OH)5.5(γ-SiW10O36)4]}·ca66H2O (U4), was synthesized using the Keggin-type precursor [γ-SiW10O36]8- and UO2(NO3)2. U4 was further characterized by single-crystal X-ray diffraction, FT-IR, Raman spectroscopy, solid-state diffuse reflection spectroscopy, ICP-OES, ESI-MS, TGA, and PXRD. The central {K2(UO2)4Cl0.5(OH)5.5} chromophore was constructed dexterously from four uranyl, four halves of K ions, 5.5 bridging µ2-OH, and disordered Cl ions, and was further stabilized by four {γ-SiW10} moieties to construct the tetramer [K2(UO2)4Cl0.5(OH)5.5(γ-SiW10O36)4]28-. Notably, U4 could work as an effective bifunctional Lewis acid-base catalyst for the synthesis of pyrazoles via the condensation of hydrazines with 1,3-diketones under mild conditions, which is attributed to the synergetic effect of the Lewis acidity of U(VI) and the Lewis basicity of {γ-SiW10}.

Generation and propagation of yeast prion [URE3] are elevated under electromagnetic field.

In this study, we studied the effect of 2.0 GHz radio frequency electromagnetic field (RF-EMF) and 50 Hz extremely low frequency electromagnetic field (ELF-EMF) exposure on prion generation and propagation using two budding yeast strains, NT64C and SB34, as model organisms. Under exposure to RF-EMF or ELF-EMF, the de novo generation and propagation of yeast prions [URE3] were elevated in both strains. The elevation increased over time, and the effects of ELF-EMF occurred in a dose-dependent manner. The transcription and expression levels of the molecular chaperones Hsp104, Hsp70-Ssa1/2, and Hsp40-Ydj1 were not statistically significantly changed after exposure. Furthermore, the levels of ROS, as well as the activities of superoxide dismutase (SOD) and catalase (CAT), were significantly elevated after short-term, but not long-term exposure. This work demonstrated for the first time that EMF exposure could elevate the de novo generation and propagation of yeast prions and supports the hypothesis that ROS may play a role in the effects of EMF on protein misfolding. The effects of EMF on protein folding and ROS levels may mediate the broad effects of EMF on cell function.

Exposure of ELF-EMF and RF-EMF Increase the Rate of Glucose Transport and TCA Cycle in Budding Yeast.

In this study, we investigated the transcriptional response to 50 Hz extremely low frequency electromagnetic field (ELF-EMF) and 2.0 GHz radio frequency electromagnetic field (RF-EMF) exposure by Illumina sequencing technology using budding yeast as the model organism. The transcription levels of 28 genes were upregulated and those of four genes were downregulated under ELF-EMF exposure, while the transcription levels of 29 genes were upregulated and those of 24 genes were downregulated under RF-EMF exposure. After validation by reverse transcription quantitative polymerase chain reaction (RT-qPCR), a concordant direction of change both in differential gene expression (DGE) and RT-qPCR was demonstrated for nine genes under ELF-EMF exposure and for 10 genes under RF-EMF exposure. The RT-qPCR results revealed that ELF-EMF and RF-EMF exposure can upregulate the expression of genes involved in glucose transportation and the tricarboxylic acid (TCA) cycle, but not the glycolysis pathway. Energy metabolism is closely related with the cell response to environmental stress including EMF exposure. Our findings may throw light on the mechanism underlying the biological effects of EMF.

Coupling Mechanism of Electromagnetic Field and Thermal Stress on Drosophila melanogaster.

Temperature is an important factor in research on the biological effects of extremely low-frequency electromagnetic field (ELF-EMF), but interactions between ELF-EMF and temperature remain unknown. The effects of ELF-EMF (50 Hz, 3 mT) on the lifespan, locomotion, heat shock response (HSR), and oxidative stress (OS) of Canton-Special (CS) and mutant w1118 flies were investigated at 25°C and 35°C (thermal stress). Results showed that thermal stress accelerated the death rates of CS and w1118 flies, shortened their lifespan, and influenced their locomotion rhythm and activity. The upregulated expression levels of heat shock protein (HSP) 22, HSP26, and HSP70 indicated that HSR was enhanced. Thermal stress-induced OS response increased malondialdehyde content, enhanced superoxide dismutase activity, and decreased reactive oxygen species level. The effects of thermal stress on the death rates, lifespan, locomotion, and HSP gene expression of flies, especially w1118 line, were also enhanced by ELF-EMF. In conclusion, thermal stress weakened the physiological function and promoted the HSR and OS of flies. ELF-EMF aggravated damages and enhanced thermal stress-induced HSP and OS response. Therefore, thermal stress and ELF-EMF elicited a synergistic effect.

Rif1 controls DNA replication by directing Protein Phosphatase 1 to reverse Cdc7-mediated phosphorylation of the MCM complex.

Initiation of eukaryotic DNA replication requires phosphorylation of the MCM complex by Dbf4-dependent kinase (DDK), composed of Cdc7 kinase and its activator, Dbf4. We report here that budding yeast Rif1 (Rap1-interacting factor 1) controls DNA replication genome-wide and describe how Rif1 opposes DDK function by directing Protein Phosphatase 1 (PP1)-mediated dephosphorylation of the MCM complex. Deleting RIF1 partially compensates for the limited DDK activity in a cdc7-1 mutant strain by allowing increased, premature phosphorylation of Mcm4. PP1 interaction motifs within the Rif1 N-terminal domain are critical for its repressive effect on replication. We confirm that Rif1 interacts with PP1 and that PP1 prevents premature Mcm4 phosphorylation. Remarkably, our results suggest that replication repression by Rif1 is itself also DDK-regulated through phosphorylation near the PP1-interacting motifs. Based on our findings, we propose that Rif1 is a novel PP1 substrate targeting subunit that counteracts DDK-mediated phosphorylation during replication. Fission yeast and mammalian Rif1 proteins have also been implicated in regulating DNA replication. Since PP1 interaction sites are evolutionarily conserved within the Rif1 sequence, it is likely that replication control by Rif1 through PP1 is a conserved mechanism.

Gene expression and reproductive abilities of male Drosophila melanogaster subjected to ELF-EMF exposure.

Extremely low frequency electromagnetic field (ELF-EMF) exposure is attracting increased attention as a possible disease-inducing factor. The in vivo effects of short-term and long-term ELF-EMF exposure on male Drosophila melanogaster were studied using transcriptomic analysis for preliminary screening and QRT-PCR for further verification. Transcriptomic analysis indicated that 439 genes were up-regulated and 874 genes were down-regulated following short-term exposures and that 514 genes were up-regulated and 1206 genes were down-regulated following long-term exposures (expression >2- or <0.5-fold, respectively). In addition, there are 238 up-regulated genes and 598 down-regulated genes in the intersection of short-term and long-term exposure (expression >2- or <0.5-fold). The DEGs (differentially expressed genes) in D. melanogaster following short-term exposures were involved in metabolic processes, cytoskeletal organization, mitotic spindle organization, cell death, protein modification and proteolysis. Long-term exposure let to changes in expression of genes involved in metabolic processes, response to stress, mitotic spindle organization, aging, cell death and cellular respiration. In the intersection of short-term and long-term exposure, a series of DEGs were related to apoptosis, aging, immunological stress and reproduction. To check the ELF-EMF effects on reproduction, some experiments on male reproduction ability were performed. Their results indicated that short-term ELF-EMF exposure may decrease the reproductive ability of males, but long-term exposures had no effect on reproductive ability. Down-regulation of ark gene in the exposed males suggests that the decrease in reproductive capacity may be induced by the effects of ELF-EMF exposure on spermatogenesis through the caspase pathway. QRT-PCR analysis confirmed that jra, ark and decay genes were down regulated in males exposed for 1 Generation (1G) and 72 h, which suggests that apoptosis may be inhibited in vivo. ELF-EMF exposure may have accelerated cell senescence, as suggested by the down-regulation of both cat and jra genes and the up-regulation of hsp22 gene. Up-regulation of totA and hsp22 genes during exposure suggests that exposed flies might induce an in vivo immune response to counter the adverse effects encountered during ELF-EMF exposure. Down-regulation of cat genes suggests that the partial oxidative protection system might be restrained, especially during short-term exposures. This study demonstrates the bioeffects of ELF-EMF exposure and provides evidence for understanding the in vivo mechanisms of ELF-EMF exposure on male D. melanogaster.

The effect of Ku on telomere replication time is mediated by telomere length but is independent of histone tail acetylation.

DNA replication in Saccharomyces cerevisiae proceeds according to a temporal program. We have investigated the role of the telomere-binding Ku complex in specifying late replication of telomere-proximal sequences. Genome-wide analysis shows that regions extending up to 80 kb from telomeres replicate abnormally early in a yku70 mutant. We find that Ku does not appear to regulate replication time by binding replication origins directly, nor is its effect on telomere replication timing mediated by histone tail acetylation. We show that Ku instead regulates replication timing through its effect on telomere length, because deletion of the telomerase regulator Pif1 largely reverses the short telomere defect of a yku70 mutant and simultaneously rescues its replication timing defect. Consistent with this conclusion, deleting the genome integrity component Elg1 partially rescued both length and replication timing of yku70 telomeres. Telomere length-mediated control of replication timing requires the TG(1-3) repeat-counting component Rif1, because a rif1 mutant replicates telomeric regions early, despite having extended TG(1-3) tracts. Overall, our results suggest that the effect of Ku on telomere replication timing results from its impact on TG(1-3) repeat length and support a model in which Rif1 measures telomere repeat length to ensure that telomere replication timing is correctly programmed.

Hsp40 interacts directly with the native state of the yeast prion protein Ure2 and inhibits formation of amyloid-like fibrils.

Ure2 is the protein determinant of the [URE3] prion phenotype in Saccharomyces cerevisiae and consists of a flexible N-terminal prion-determining domain and a globular C-terminal glutathione transferase-like domain. Overexpression of the type I Hsp40 member Ydj1 in yeast cells has been found to result in the loss of [URE3]. However, the mechanism of prion curing by Ydj1 remains unclear. Here we tested the effect of overexpression of Hsp40 members Ydj1, Sis1, and Apj1 and also Hsp70 co-chaperones Cpr7, Cns1, Sti1, and Fes1 in vivo and found that only Ydj1 showed a strong curing effect on [URE3]. We also investigated the interaction of Ydj1 with Ure2 in vitro. We found that Ydj1 was able to suppress formation of amyloid-like fibrils of Ure2 by delaying the process of fibril formation, as monitored by thioflavin T binding and atomic force microscopy imaging. Controls using bovine serum albumin, Sis1, or the human Hsp40 homologues Hdj1 or Hdj2 showed no significant inhibitory effect. Ydj1 was only effective when added during the lag phase of fibril formation, suggesting that it interacts with Ure2 at an early stage in fibril formation and delays the nucleation process. Using surface plasmon resonance and size exclusion chromatography, we demonstrated a direct interaction between Ydj1 and both wild type and N-terminally truncated Ure2. In contrast, Hdj2, which did not suppress fibril formation, did not show this interaction. The results suggest that Ydj1 inhibits Ure2 fibril formation by binding to the native state of Ure2, thus delaying the onset of oligomerization.

The yeast prion protein Ure2: structure, function and folding.

The Saccharomyces cerevisiae protein Ure2 functions as a regulator of nitrogen metabolism and as a glutathione-dependent peroxidase. Ure2 also has the characteristics of a prion, in that it can undergo a heritable conformational change to an aggregated state; the prion form of Ure2 loses the regulatory function, but the enzymatic function appears to be maintained. A number of factors are found to affect the prion properties of Ure2, including mutation and expression levels of molecular chaperones, and the effect of these factors on structure and stability are being investigated. The relationship between structure, function and folding for the yeast prion Ure2 are discussed.