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Background Bacterially-produced recombinant prion protein (rPrP) has traditionally been used for in vitro fibrillation assays and reagent development for prion disease research. In recent years, it has also been used as a substrate for real-time quaking-induced conversion (RT-QuIC), a very sensitive method of detecting the presence of the misfolded, disease-associated isoform of the prion protein (PrP d ). Multi-centre trials have demonstrated that RT-QuIC is a suitably reliable and robust technique for clinical practice; however, in the absence of a commercial supplier of rPrP as a substrate for RT-QuIC, laboratories have been required to independently generate this key component of the assay. No harmonized method for producing the protein has been agreed upon, in part due to the variety of substrates that have been applied in RT-QuIC. Methods This study examines the effects of two different rPrP refolding protocols on the production, QuIC performance, and structure characteristics of two constructs of rPrP commonly used in QuIC: full length hamster and a sheep-hamster chimeric rPrP. Results Under the described conditions, the best performing substrate was the chimeric sheep-hamster rPrP produced by shorter guanidine-HCl exposure and faster gradient elution. Conclusions The observation that different rPrP production protocols influence QuIC performance indicates that caution should be exercised when comparing inter-laboratory QuIC results.

This study examined the extent to which a sample of 804 undergraduates at a large southeastern university used communication technology (e.g., cell phone, email, social network sites) to monitor or control partners in intimate relationships and to evaluate their perceptions of the appropriateness of these behaviors. Results of the online survey revealed that half of both female and male respondents reported the use of communication technology to monitor partners, either as the initiator or victim. Females were significantly more likely than males to monitor the email accounts of their partners (25% vs. 6%) and to regard doing so as appropriate behavior. Limitations and implications are suggested.

Previously, it has been demonstrated that an "adaptive response" that includes the prevention, repair, and removal of oxidative damage can be evoked by radiation at dose rates substantially lower than those at which risks have been observed. The exact pathogenic mechanism of prion diseases is unknown, but circumstantial evidence suggests that oxidative stress plays a central role. Exposure of prion-infected mice to four 500 mGy/fraction doses of 60Co γ-radiation administered every other day at a low dose rate (0.5 mGy/min) starting at 2 days before infection, 7 days postinfection (dpi), or 50 dpi significantly prolonged the survival of infected mice. The 500-mGy radiation treatments started at 50 dpi also significantly prolonged the symptom-free period of the disease and caused a significant delay in the rise of the 8-hydroxydeoxyguanosine concentration observed in the urine of nonirradiated infected mice at 98 dpi. The duration of the reduction in oxidative stress achieved by the radiation treatments was similar in length to the prolonged survival of the irradiated mice. This suggests that the adaptive response induced by low-dose whole-body radiation treatments prolongs the survival of prion-infected mice by reducing oxidative stress.

S-Adenosylmethionine (AdoMet) is the methyl donor of numerous methylation reactions. The current model is that an increased concentration of AdoMet stimulates DNA methyltransferase reactions, triggering hypermethylation and protecting the genome against global hypomethylation, a hallmark of cancer. Using an assay of active demethylation in HEK 293 cells, we show that AdoMet inhibits active demethylation and expression of an ectopically methylated CMV-GFP (green fluorescent protein) plasmid in a dose-dependent manner. The inhibition of GFP expression is specific to methylated GFP; AdoMet does not inhibit an identical but unmethylated CMV-GFP plasmid. S-Adenosylhomocysteine (AdoHcy), the product of methyltransferase reactions utilizing AdoMet does not inhibit demethylation or expression of CMV-GFP. In vitro, AdoMet but not AdoHcy inhibits methylated DNA-binding protein 2/DNA demethylase as well as endogenous demethylase activity extracted from HEK 293, suggesting that AdoMet directly inhibits demethylase activity, and that the methyl residue on AdoMet is required for its interaction with demethylase. Taken together, our data support an alternative mechanism of action for AdoMet as an inhibitor of intracellular demethylase activity, which results in hypermethylation of DNA.

Previous lines of evidence have shown that inhibition of DNA methyltransferase (MeTase) can arrest tumor cell growth; however, the mechanisms involved were not clear. In this manuscript we show that out of 16 known tumor suppressors and cell cycle regulators, the cyclin-dependent kinase inhibitor p21 is the only tumor suppressor induced in the human lung cancer cell line, A549, following inhibition of DNA MeTase by a novel DNA MeTase antagonist or antisense oligonucleotides. The rapid induction of p21 expression points to a mechanism that does not involve demethylation of p21 promoter. Consistent with this hypothesis, we show that part of the CpG island upstream of the endogenous p21 gene is unmethylated and that the expression of unmethylated p21 promoter luciferase reporter constructs is induced following inhibition of DNA MeTase. These results are consistent with the hypothesis that the level of DNA MeTase in a cell can control the expression of a nodal tumor suppressor by a mechanism that does not involve DNA methylation.

Vertebrates have developed multiple mechanisms to coordinate the replication of epigenetic and genetic information. Dnmt1 encodes the maintenance enzyme DNA-methyltransferase, which is responsible for propagating the DNA methylation pattern and the epigenetic information that it encodes during replication. Direct sequence analysis and bisulfite mapping of the 5' region of DNA-methyltransferase 1 (dnmt1) have indicated the presence of many sequence elements associated with previously characterized origins of DNA replication. This study tests the hypothesis that the dnmt1 region containing these elements is an origin of replication in human cells. First, we demonstrate that a vector containing this dnmt1 sequence is able to support autonomous replication when transfected into HeLa cells. Second, using a gel retardation assay, we show that it contains a site for binding of origin-rich sequences binding activity, a recently purified replication protein. Finally, using competitive polymerase chain reaction, we show that replication initiates in this region in vivo. Based on these lines of evidence, we propose that initiation sites for DNA replication are located between the first intron and exon 7 of the human dnmt1 locus.

The study of the biological role of DNA methyltransferase (DNA MeTase) has been impeded by the lack of direct and specific inhibitors. This report describes the design of potent DNA based antagonists of DNA MeTase and their utilization to define the interactions of DNA MeTase with its substrate and to study its biological role. We demonstrate that the size, secondary structure, hemimethylation, and phosphorothioate modification strongly affect the antagonists interaction with DNA MeTase whereas base substitutions do not have a significant effect. To study whether DNA MeTase is critical for cellular transformation, human lung non-small carcinoma cells were treated with the DNA MeTase antagonists. Ex vivo, hairpin inhibitors of DNA MeTase are localized to the cell nucleus in lung cancer cells. They inhibit DNA MeTase, cell growth, and anchorage independent growth (an indicator of tumorigenesis in cell culture) in a dose-dependent manner. The inhibitors developed in this study are the first documented example of direct inhibitors of DNA MeTase in living cells and of modified oligonucleotides as bona fide antagonists of critical cellular proteins.

There is a great variety of retirement income systems around the world, but which ones are producing good outcomes? Which ones are sustainable into the future? The Melbourne Mercer Global Pension Index considers more than forty indicators in calculating an index value for the systems in eleven countries spread over five continents. In this first-ever broad-based comparison, none of the eleven countries receive an ‘A’ grade. At the other end of the quality spectrum, none receive a failing ‘E’ either. However, the actual grade range from ‘B’ to ‘D’ reflects material differences in the quality of the eleven retirement income systems as measured by the Index.

Endonuclease analysis of mitochondrial DNA from wild populations of Norwegian and Scottish Atlantic salmon has identified an Hae III restriction fragment length polymorphism which is unique to the Norwegian fish tested. This genetic variant may have potential for use as a population marker for studies of genetic mixing of escaped farm salmon of Norwegian origin with wild populations in Scotland.

Purified diets with copper levels of 2.5 and 500 mg/kg and dietary zinc levels ranging from 34 to 1000 mg/kg were fed for 20 weeks to rainbow trout of mean initial weight 8 g. Growth and feed conversion were similar in all groups of fish and no gross pathologies were observed. Liver copper levels were greatest in trout given the high copper diets; increasing dietary zinc intake reduced these hepatic copper concentrations. Subcellular fractionation of the livers showed that in all groups of trout 70–80% of the hepatic copper was present in the nuclear and mitochondrial fractions. In contrast, the cytosol and microsomal fractions contained, on average, 73% of the liver zinc. Total liver superoxide dismutase activity was constant in all groups of fish. However, in trout fed the high copper diets the relative proportions of the copper-zinc and the manganese superoxide dismutase were correlated with the dietary zinc intake. Increasing dietary zinc reduced the activity of the manganese enzyme and increased that of the copper-zinc metalloenzyme.

Flexible, self-organised work groups will become increasingly important in rich countries like the U.S. as employment in the formal economic system continues to decline. Educational models and methods based on the linking of learning with productive work developed in Third World countries can give a valuable stimulus to educational innovation in those industrialized countries hardest hit by the current crisis. Education with production will be important in equipping people for the new situation. The article discusses examples of several types of education with production. Among issues raised are: what skills are needed; what sorts of people the different types of post-school education with production cater for; how to extend the benefits of self-organised work; some important factors in introducing education with production at school level, including demands on the teacher and relevance to pupils' needs. The role of the Foundation for Education with Production is briefly described.

Purified diets with 15 and 150 mg supplemental copper/kg with dietary copper: zinc ratios of 1:1 and 1:4 were fed for 20 weeks to rainbow trout of mean initial weight 15 g. No gross pathologies were noted in any group of fish; growth and feed conversion were the same in all groups of trout. Plasma zinc levels were positively correlated with dietary zinc intake but dietary copper level had no effect on plasma copper. Hepatic levels of copper and zinc were also related to the dietary intake of the respective minerals. The dietary copper: zinc ratio caused some small changes in the plasma and hepatic levels of a few minerals, but no evidence was found to suggest any zinc—copper antagonism in rainbow trout. The activity of the copper, zinc metalloenzyme superoxide dismutase in liver was unaffected by dietary copper or zinc intake.